Expression vectors for producing modified proteins

ABSTRACT

Modified proteins, modified interferons α&#39;s and β&#39;s, phosphorylated modified proteins and DNA sequences encoding the above, applications and uses thereof. Modified phosphorylated Hu-IFN-α-like proteins are provided which carry an identifiable label such as a radio-label. Corresponding phosphorylatable Hu-IFN-α-like proteins which contain a putative phosphorylation site. DNA sequences which encode a Hu-IFN-α-like protein and contain a sequence encoding a putative phosphorylatable site. Appropriate expression vectors are used to transform compatible host cells of various microorganisms, such as  E. coli . Numerous uses for the phosphorylated proteins are disclosed.

This application is a continuation of application Ser. No. 08/487,057,filed Jun. 7, 1995, now U.S. Pat. No. 6,150,503, which is a continuationof application Ser. No. 08/375,112 filed Jan. 19, 1995, now U.S. Pat.No. 6,225,455, which is a continuation of application Ser. No.08/156,875, filed Nov. 23, 1993, now abandoned, which is a continuationof application Ser. No. 07/264,271, filed Oct. 28, 1988, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of recombinant DNAtechnology, to means, methods of utilizing this technology to synthesizeuseful functional proteins or polypeptides which include one or morephosphate (or thiophosphate) groups which are radio-labelled, to theseand various other products useful in biomedical, medical, biochemicalapplications including diagnostics, prophylatics and therapeutics.

More specifically the invention relates to new interferons, especiallyto leukocyte, or alpha interferon(s) and fibroblast or beta interferonwhich contain one or more radioactive phosphorylated groups, to DNAsequences encoding putative phosphorylatable sites, which code for thesenew interferons.

BACKGROUND OF THE INVENTION

Radio-labelled proteins have numerous medical, biological, clinical,scientific and other applications. Interferons, specifically, labelledwith ¹²⁵I have been used for binding and crosslinking studies (1, 17,27-32, 34-37).¹ Human IFN-α's, -β, and -gamma have all beenradio-iodinated by various procedures (reviewed in Pestka et al (2)).However, proteins labelled with radioactive iodine have seriouswell-known disadvantages and hazards.

¹The scientific publications, patents or other literature(“publications”) to which reference is made herein are referenced bynumerals and identified further towards the end of this text. All ofthese publications are incorporated herein by reference.

The study of cell surface receptors for the interferons requiresradio-labelled interferons, such as interferons labelled with ¹²⁵I, withhigh biological and high radio-specific activity. Several years ago, itwas found that interferon gamma² can be phosphorylated to very highradio-specific activity while retaining biological activity (3, 4).Thus, [³²P]Hu- and Mu-IFN-gamma were used for studying the human andmurine IFN-gamma receptors, respectively (5, 6, 9). These studies werecarried out by phosphorylating human and murine interferon gamma (Hu-and Mu-IFN-gamma) with cyclic AMP-dependent protein kinase from bovineheart muscle and [gamma-³²P]ATP (3). These phosphorylated and³²P-labelled interferons have provided valuable reagents (3, 4) of highradio-specificity to study cell surface receptors (5, 6) and to identifythe chromosome encoding the gene for Hu-IFN-gamma (7, 8) andMu-IFN-gamma (9) receptors. For all of these studies and applications,interferons which are phosphorylated are most useful. Several reportsidentified the phosphorylation sites of Hu- and Mu-IFN-gamma as serineresidues near the COOH termini (4, 5, 10, 11).

²The abbreviations used have followed standard nomenclature as describedin detail in Methods of Enzymology, Interferons, Vol. 119, Part C,Edited by Sidney Pestka, Section I, Introduction (Reference 25). Inbrief, interferon alpha, beta, and gamma are designated IFN-α, IFN-β,and -gamma, respectively. The species of origin is designated by aprefix Hu, Mu, Bo, etc. for human, murine, or bovine species,respectively, as Hu-IFN-α, Hu-IFN-β, or Hu-IFN-gamma, for example.

However, under conditions used for the phosphorylation of IFN-gamma, itwas reported that Hu-IFN-αA and Hu-IFN-β cannot be phosphorylated by thecyclic AMP (cAMP)-dependent protein kinase (2, 3). A review of thephosphorylation of the various classes or groups of interferons andother proteins (1, 3, 4, 20, 21, 22, 38, 39, 40, 64) confirms thatresearchers have not successfully phosphorylated Hu-IFN-α or Hu-IFN-βunder conditions under which gamma interferons have been phosphorylated.It has been reported indeed that recombinant IFN-α and IFN-β were notphosphorylated (3) and as a consequence it was uncertain whether anavailable site was present.

In the light of problems with iodinated compounds and limitations foruse of iodinated IFN-gamma, it is understandable that there is a keeninterest and need in making available phosphorylated Hu-IFN-α and -βwhich can be labelled for numerous practical, scientific and commercialapplications.

Likewise, there is such interest and need for other phosphorylated—andlabelled—polypeptides which are not available yet in such chemicalconfigurations. For example, a phosphorylatable tumor necrosis factor(TNF) would be valuable to study the receptor for TNF. TNF is notphosphorylatable with the cAMP-dependent bovine heart kinase. Indeed, ithas been reported that interest in protein phosphorylation has increasedenormously over the past few years (38, 39).

The invention as will be described in detail hereinafter contributes tomeeting these and other needs.

By way of further background to the invention, the term “interferon”describes a family of animal proteins which possess antiviral,antiproliferative and other potentially useful properties. There appearto be three major classes of interferons: leukocyte (or alphainterferon), fibroblast (or beta interferon) and immune (or gammainterferon) (1, 2). Detailed description of interferons is found invarious publications including in references 1, 2, U.S. Pat. Nos.4,727,138; 4,734,491; 4,737,462, and many others; various hybrid humanleukocyte interferons are described in U.S. Pat. No. 4,414,150 and inreference 46. In general the standard class of human IFN-α's arepolypeptides of 165-166 amino acids (see reference 1 for details ofhuman and non-human interferon-a species); some species have beenisolated that lack the 10 COOH-terminal amino acid residues; and somespecies of IFN-α are glycosylated. The amino acid sequences of Hu-IFN-αspecies and of Hu-IFN-β derived from cNDA or genomic DNA sequences aredescribed in (1, Section I). Recombinant DNA-derived interferonsincluding Hu-IFN-α, -β, and -gamma and corresponding interferons fromother animal species are likewise well described (1, 2). Variousmodifications of human and murine interferons have been reported. Newnon-natural human and murine interferons with often markedly changedbiological properties have been constructed (1, 24, 45). The terminology“non-natural” is a term of art which refers to recombinant DNAinterferons obtained by altering the nucleotide sequence of coding cDNAs(45).

The term “Hu-IFN-α” as used herein is intended to include all differentspecies of alpha interferons. A large number of DNA sequencescorresponding to the interferons from various species have been isolatedand identified. Likewise various IFN-βs and IFN-gamma(s) are disclosed.The invention encompasses all of these members of the family (reference1, pages 5-14).

The term “native” as used herein refers to the proteins, e.g.,interferons, which proteins are naturally produced; “synthetic” and“non-natural” refers to proteins produced by synthetic orDNA-recombinant procedures, either type which do not contain aphosphorylatable site (or where the phosphorylatable site isinaccessible, for instance due to the configuration of the protein),which protein in accordance with the invention is to be phosphorylated.

This invention contemplates and includes all interferons native,natural, modified, or recombinant DNA interferon-like proteins which aremodifiable by introduction of one or more phosphate or analog groups.All of these interferons and others known in the art or to be known arewithin the contemplation of the invention. The present invention isprincipally concerned with various modified proteins or polypeptides,and alpha and beta interferons.

When reference is made to IFN-alpha, the term is intended to cover andinclude the various alpha species.

The term “modified” is used in this invention broadly, and means forinstance, when reference is made to proteins, a protein which has beenprovided with a phosphorylatable site or provided with a phosphoruslabel (or analog label). The nucleotide sequences which code for suchamino acid sequences which contain a putative phosphorylation site arealso designated as “modified”, when appropriate.

The term “unphosphorylatable” protein means a protein which normally hasnot been phosphorylatable (or phosphorylated) for whatever reason, e.g.,either because it does not contain a putative phosphorylatable site andcorrespondingly, the DNA sequence which codes for the protein does notcontain the DNA sequence coding for the putative amino acid recognitionsequence; or because such site is not accessible for phosphorylation.

The term “provided with” or “having provision(s) for” or liketerminology is used in this invention broadly, and means both “fused”and “inserted”. Illustrative are the hybrid-fused Hu-IFN-αA/gamma(illustrated in FIG. 1) and Hu-IFN-αA-P1, -P2 and -P3 (illustrated inFIG. 8), respectively. Thus, the nucleotide insert can be within thecoding region of the gene at one end thereof or anywhere within thecoding region. These variants are all considered to be within the term“modified,” which can refer to the amino acid sequence or to thenucleotide sequences, as will become apparent from the descriptionhereinafter.

The term “comprises” or “comprising” covers and includes all situationsregardless where the amino acid recognition sequence (or the nucleotidesequence coding for it) is located.

By way of further background in the preferred method of the invention,phosphorylation is carried out by means of a protein kinase. Proteinkinases catalyze the transfer of the gamma phosphoryl group of ATP to anacceptor protein substrate. However, as described herein the inventionis not limited to kinases for which the acceptor site is a particularamino acid (like serine) but includes also those for which the site isanother amino acid in the sequence, and in general includes proteinkinases as a whole.

The term “protein” (or polypeptide) as used herein is intended toinclude glycoproteins (as well as proteins having other additions). Acase in point is that of natural Hu-IFN-β which has been shown to be aglycoprotein; when produced in E. coli by recombinant DNA techniques,Hu-IFN-β is not glycosylated. Glycosylated interferons have beenreported to be obtained by expressing the proteins in animal cells or inyeast (as is discussed in reference 1 at pps. 383-433 and 453-464; andin references 48-55, 84-92).

The term “biological activities” or like terms as used herein inconjunction with proteins is intended to be interpreted broadly. In thecase of the interferon-like proteins, it includes all known (or to bediscovered) properties including properties specific to Hu-IFN-α's or toHu-IFN-β or common to both, such as their antiviral activity and theircapability to modulate antigens of the major histocompatibility complex(MHC), in particular to induce an increase in surface expression ofclass I MHC antigens, including β₂-macroglobulin.

“Functional” proteins are proteins which have a biological or otheractivity or use.

The term “active areas” or “biologically active” areas or segments orequivalent terminology often refers to the presence of a particularconformation or folding of the protein molecule, or for instance, tospecific disulfide bridges between specific amino acids in the sequence,but of course is not limited thereto.

The term “vector” as used herein means a plasmid, a phage DNA, or otherDNA sequence that (1) is able to replicate in a host cell, (2) is ableto transform a host cell, and (3) contains a marker suitable foridentifying transformed cells.

Throughout the description of the invention and the claims, andfollowing convention, the “singular” includes the “plural”; forinstance, a phosphorylatable or phosphorylation site, means at least onesuch site, unless indicated otherwise.

Other terminology used herein will become apparent from the descriptionwhich follows.

BRIEF DESCRIPTION OF THE PRIOR ART

Background references for the subject invention are referred to withinthe body and towards the end of the text.

As representative of United States patents which relate to interferon,the following may be mentioned:

U.S. Pat. No. 4,503,035 to Pestka et al relates to human leukocyteinterferon as a homogeneous protein species, such as species α₂, α₂, andβ₁, and others. For a discussion of terminology of natural andrecombinant interferons see references 1 (pps. 3-23), 24, 102, and 103(footnote p. 112 and text);

U.S. Pat. No. 4,748,233 to Sloma relates to a cloned human alphainterferon GX-1 gene which specifies the synthesis of alpha interferonGX-1;

U.S. Pat. No. 4,746,608 to Mizukami et al relates to a process forproducing peptides generally such as interferon and in particular betainterferon with microorganisms containing recombinant DNA;

U.S. Pat. No. 4,738,931 to Sugano et al relates to a DNA sequencecontaining a human interferon-β gene and the production of humaninterferon-β in eukaryotes;

U.S. Pat. No. 4,738,921 to Belagaje et al relates to a recombinant DNAexpression vector and a process for producing peptides generallyincluding interferon. The recombinant DNA vector comprises a derivativeof the tryptophan promoter-operator-leader sequence useful for theexpression;

U.S. Pat. No. 4,737,462 to Mark et al relates to modified interferon-βwherein the cysteine residue at position 17 is substituted by serine. Inconnection with that patent, it is interesting to note that the Serwhich is provided in replacement of the Cys 17 does not constitute partof the amino acid sequence recognizable by the cAMP-dependent kinase, asdescribed in connection with the present invention;

U.S. Pat. No. 4,734,491 to Caruthers relates to a DNA sequence and amethod for the construction of recombinant DNA sequences which encodehybrid lymphoblastoid-leukocyte human interferons which have biologicalor immunological activity;

U.S. Pat. No. 4,727,138 to Goeddel et al relates to recombinant DNA forencoding polypeptides specifically human immune interferon (interferongamma);

U.S. Pat. No. 4,705,750 to Nasakazu et al relates to recombinant DNAhaving promoter activity and a process for the production of peptidesincluding human immune interferon by a transformed bacillus;

U.S. Pat. No. 4,681,931 to Obermeier et al relates to a process for theisolation and purification of alpha interferons;

U.S. Pat. No. 4,659,570 to Terano relates to a stabilizedphysiologically active polypeptide especially gamma interferon;

U.S. Pat. No. 4,559,302 to Ingolia relates to DNA sequences which encodevarious functional polypeptides including human interferon;

U.S. Pat. No. 4,559,300 to Kovacevic et al relates to a method forproducing functional polypeptides including human interferon in astreptomyces host cell and transformed bacillus;

U.S. Pat. No. 4,530,904 to Hershberger et al relates to a method forprotecting a bacterium transformed with recombinant DNA that can producefunctional polypeptides such as human interferon and non-humaninterferon from bacteriophage activity;

U.S. Pat. No. 4,506,013 to Hershberger et al relates to a method forstabilizing and selecting recombinant DNA host cells which producefunctional polypeptides generally including human and non-humaninterferon, and the transformed host cells;

U.S. Pat. No. 4,436,815 to Hershberger et al relates to a similar methodand product;

U.S. Pat. No. 4,420,398 to Castino relates to a purification method forhuman interferon;

U.S. Pat. No. 4,262,090 to Colby, Jr. et al relates to a method forproducing mRNA for mammalian interferon;

U.S. Pat. No. 4,751,077 to Bell et al relates to a modified humaninterferon-beta in which tyrosine is replaced by cysteine. The modifiedinterferon has improved stability;

U.S. Pat. No. 4,748,234 to Dorin et al relates to a process forrecovering and removing biologically active proteins specifically humaninterferon-β from a genetically engineered host microorganism cell;

U.S. Pat. No. 4,748,119 to Rich et al relates to a process of in vitrosite-directed mutagenesis or DNA deletion/substitution of DNA segmentswhich results in DNA segments capable of enhanced expression andproduction of polypeptides in general including interferons;

U.S. Pat. No. 4,745,057 to Beckage et al relates to a process in whichtransformed yeast cells express biologically-active polypeptides ingeneral including human and non-human interferon;

U.S. Pat. No. 4,745,053 to Mitsuhashi relates to a process for inducingthe production of human interferon from whole blood and for measuringblood interferon productivity level and a clinical assay for cancer;

U.S. Pat. No. 4,743,445 to Delwiche et al relates to a method fortreating (hemorrhagic) thrombocythemia by using alpha-type interferons;

U.S. Pat. No. 4,741,901 to Levinson et al relates to recombinant DNAtechnology to produce polypeptides generally including human fibroblastand human and hybrid leukocyte interferons;

U.S. Pat. No. 4,738,928 to Weissmann et al relates to a method foridentifying and isolating a recombinant DNA segment coding for apolypeptide, and cloning the said DNA segment.

It is noteworthy that the above reviewed patent literature does notaddress or disclose human interferons which have phosphorylated groups(or isotopes thereof).

SUMMARY AND GENERAL CONCEPTS OF THE INVENTION

In a broad sense, the invention contemplates labellable and labelledproteins, e.g. radio-labellable and radio-labelled proteins, and DNA andcNDA molecules encoding the radio-labellable proteins.

The invention encompasses recombinant DNA sequences which encodefunctional proteins having one or more putative phosphorylation sites;expression vectors for expressing the functional protein; transformedhost, methods of expressing the modified proteins and the modifiedproteins.

In one embodiment, the invention provides radioactive-labelled humaninterferons and labelled proteins; phosphorylatable modified Hu-IFN-α(Hu-IFN-αA-P) which can be phosphorylated to high radio-specificactivity with retention of biological activity; other human interferonsmodified with various isotopes of phosphorus (e.g., ³²P, ³²P), or withsulfur (e.g., ³⁵S, ³⁶S); labelled proteins with phosphorus or analogs.In accordance with the invention, the human interferons and modifiedproteins may have single or multiple radioactive labels.

The invention also provides such interferons and proteins made byrecombinant DNA techniques, including the Hu-IFN-αA-P human interferonsradio-labelled with phosphorus or with sulfur, and recombinantDNA-produced radio-labelled polypeptides and proteins.

The invention further provides DNA sequences encoding a functionalprotein which possesses one or more labelling sites and is sufficientlyduplicative of human interferons for the protein sequences to possess atleast one of the biological properties of interferons (like antiviral,cell growth inhibiting, and immunomodulatory properties). Further, thereis provided a recombinant-DNA containing a coding sequence for aputative recognition site for a kinase; the recombinant expressionvector; the host organisms transformed with the expression vector thatincludes the DNA sequence and an expressed modified protein. In theinvention, there is used a method involving site-specific mutagenesisfor constructing the appropriate expression vector, a host transformedwith the vector and expressing the modified proteins, in particular themodified human interferons.

The invention provides in one of its several embodiments DNA sequenceswhich encode one or more putative phosphorylation sites, which sequencesencode functional proteins each of which possesses at least one putativephosphorylation site and each of which possesses at least one of thebiological properties of Hu-IFN-α or -β; also expression vectors forexpression of the functional modified Hu-IFN-α or -β under the controlof a suitable promoter such as the lambda P_(L) promoter or othersdescribed hereinafter; also the biologically active phosphorylatedHu-IFN-α and -β.

Several interesting and useful applications of these modified humaninterferons and proteins are also disclosed by the description.

The invention also contemplates interferons or proteins other than theHu-IFN-α or -β, which are modified by addition of phosphorylation siteswhich allow for and are labelled to higher radio-specific activitiesthan the corresponding interferons with a single phosphorylation site.By “addition” of phosphorylation sites, there is also intended inaccordance with the invention, to include interferons or proteins inwhich a phosphorylation site heretofore unavailable or inaccessible, hasbeen modified to make the phosphorylation site available.

The invention further contemplates interferons, especially Hu-IFN-β,phosphorylated by appropriate kinases on amino acid residues other thanon the serine residue, like on threonine and/or tyrosine residues, andthe DNA sequences which code for one or more putative phosphorylationsites, which sequences code for these interferons.

In accordance with the invention, it is sufficient that a portion of thephosphorylation recognition sequence, as opposed to the entire sequence,be added when the natural protein sequence contains the remaining (orother complementary) amino acids of said recognition sequence (e.g.,Arg-Arg-Ala-Ser). In such embodiment of the invention, from 1 through 4amino acids of the sequence (in the case of Arg-Arg-Ala-Ser-Val) can besupplied to the protein, thereby constituting the complete, necessaryand Ser-containing recognition sequence. An illustration can be observedin a comparison between species Hu-IFN-αA-P1 and -P2 (in FIG. 8),wherein the natural interferon sequence contributes one Arg to thephosphorylation recognition sequence in Hu-IFN-αA-P2 when constructed inaccordance with the invention.

In Hu-IFN-αA-P3, a coding sequence (and thus an additional amino acidsequence) has been supplied with the nucleotide sequence coding for therecognition sequence positioned downstream of the natural sequencecoding for Hu-IFN-αA. Thus, Hu-IFN-αA-P3 is an illustration where anadditional amino acid sequence is positioned between the recognitionsequence and the natural amino acid sequence of Hu-IFN-αA.

This illustrates the versatility of the invention for positioning thenucleotide sequence which encodes the amino acid recognition sequencecontaining a putative phosphorylation site.

Thus, in accordance with the invention, there is constructed anucleotide sequence that codes for the necessary number and specificamino acids required for creating the putative phosphorylation site.

From the above observation, the same principles are applicable toconstruct any amino acid sequences other than the particular amino acidrecognition sequence illustrated above.

In the situations where the phosphorylation site is other than serine(as illustrated above), the DNA sequence codes for part or all of theappropriate amino acid sequence containing the putative recognition sitecontaining threonine, tyrosine, etc. Thus, where in any particularprotein one or more amino acids (at any position of the amino acidsequence) are the same as that of an amino acid recognition sequence fora kinase, it is sufficient to add (or modify) those complementary aminoacids of the amino acid recognition sequence to complete that sequence.This is accomplished by constructing a DNA sequence which codes for thedesired amino acid sequence. There may indeed be situations where suchaddition (or modification) is a more desirable procedure as where it isimportant to retain the integrity of the protein molecule to be modified(for instance, to minimize risks of affecting a particular activity,e.g., biological), or for simplicity of the genetic manipulations, orbecause either or both termini or other positions are more accessible.

The kinase recognition sequence may be positioned at either termini orother position of the DNA coding sequence, irrespective of the specificphosphorylated amino acid.

In accordance with the invention, phosphorylation of thephosphorylatable site of the protein can be performed by any suitablephosphorylation means. Phosphorylation and dephosphorylation of proteinscatalyzed by protein kinases and protein phosphatases is known to affecta vast array of proteins (21). A large number of protein kinases havebeen described (20, 21, 22, 38, 39, 47, 64, 100, 101, 108-112) and areavailable to one skilled in the art for use in the invention. Suchprotein kinases may be divided into two major groups: those thatcatalyze the phosphorylation of serine and/or threonine residues inproteins and peptides and those that catalyze the phosphorylation oftyrosine residues (see 21, 22, 38, 64, 108, for example). These twomajor categories can be subdivided into additional groups. For example,the serine/threonine protein kinases can be subdivided into cyclic AMP(cAMP)-dependent protein kinases, cyclic GMP (cGMP)-dependent kinases,and cyclic nucleotide-independent protein kinases. The recognition sitesfor many of the protein kinases have been deduced (21, 22, 38, 64, 111present illustrative examples).

In short synthetic peptides cAMP-dependent protein kinase recognize thesequence Arg-Arg-Xxx-Ser-Xxx, where Xxx represents an amino acid (21).As noted above, the cAMP-dependent protein kinase recognizes the aminoacid sequence Arg-Arg-Xxx-Ser-Xxx (21), but also can recognize someother specific sequences such as Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val (111).Many other protein serine/threonine kinases have been reported (21, 100,101, 108-112) such as glycogen synthase kinase, phosphorylase kinase,casein kinases I and II, pyruvate dehydrogenase kinase, protein kinaseC, and myosin light chain kinase.

Protein kinases which phosphorylate and exhibit specificity for tyrosine(rather than for serine, threonine, or hydroxyproline) in peptidesubstrates are the protein tyrosine kinases (PTK). Such PTKs aredescribed in the literature (22, 64). The PTKs are another class ofkinases available for use in the invention.

Another available class of kinases are the cyclic GMP-dependent(cGMP-dependent) protein kinases. The cGMP-dependent protein kinasesexhibit substrate specificity similar to, but not identical to thespecificity exhibited by cAMP-dependent protein kinases. The peptideArg-Lys-Arg-Ser-Arg-Lys-Glu was phosphorylated at serine by thecGMP-dependent protein kinase better than by the cAMP-dependent proteinkinase (21, 22, 113). It has also been shown that the cAMP-dependentprotein kinase can phosphorylate hydroxyproline in the synthetic peptideLeu-Arg-Arg-Ala-Hyp-Leu-Gly (114).

Casein kinases, widely distributed among eukaryotic organisms andpreferentially utilizing acidic proteins such as casein as substrates,have been classified into two groups, casein kinases I and II (21).Casein kinase II phosphorylated the synthetic peptideSer-Glu-Glu-Glu-Glu-Glu (115). Evaluation of results with syntheticpeptides and natural protein substrates revealed that a relatively shortsequence of amino acids surrounding the phosphate acceptor site providesthe basis for the specificity of casein kinase II (118). Accordingly,the acidic residues at positions 3 and 5 to the carboxyl-terminal sideof the serine seem to be the most important. Serine was preferentiallyphosphorylated compared to threonine. In another study (117), thepeptide Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu was found to be aspecific substrate for casein kinase II; however,Arg-Arg-Arg-Glu-Glu-Glu-Ser-Glu-Glu-Glu was a better substrate (118);and Arg-Arg-Arg-Asp-Asp-Asp-Ser-Asp-Asp-Asp was a better substrate thanArg-Arg-Arg-Glu-Glu-Glu-Ser-Glu-Glu-Glu. Thus, aspartate is preferredover glutamate (118). Acidic residues on the COOH-terminal side of theserine (threonine) are as far as known today absolutely required; acidicresidues on the amino-terminal side of the serine (threonine) enhancephosphorylation, but are not absolutely required: thus,Ala-Ala-Ala-Ala-Ala-Ala-Ser(Thr)-Glu-Glu-Glu served as a substrate forcasein kinase II, but was less effective thanAla-Ala-Ala-Glu-Glu-Glu-Ser(Thr)-Glu-Glu-Glu (118) (thedesignationSer(Thr) means serine or threonine). Casein kinases I and IIphosphorylate many of the same substrates (21) although casein kinase Idid not phosphorylate any of the decamer peptide substrates noted here(118). It was concluded from studies with a variety of syntheticpeptides that the sequence Ser-Xxx-Xxx-Glu (and by inferenceSer-Xxx-Xxx-Asp) may represent one class of sequences that fulfill theminimal requirements for recognition by casein kinase II although someother peptides and sequences may also suffice (see 118 for a detaileddiscussion).

As noted above, other kinases have been described. The mitogen-activatedS6 kinase phosphorylates the synthetic peptideArg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (109) as does a protease-activatedkinase from liver (21, 109). The rhodopsin kinase catalyzes thephosphorylation of the peptide Thr-Glu-Thr-Ser-Gln-val-Ala-Pro-Ala (21).Other protein serine/threonine kinases have been described and theirsites of phosphorylation elucidated (21).

The substrate specificity of tyrosine kinases have also been reported(64, pages 920-921; 110). A variety of synthetic or natural peptidesubstrates have been described (64, 110).

Thus, one skilled in the art has quite an adequate selection ofavailable kinases for use in the invention, which have relatively highspecificity with respect to the recognition process, but someflexibility to the specific sequence of the amino acid recognition site.Such kinases provide means for phosphorylation of putativephosphorylation sites in the desired proteins.

The selection of the position of the molecule best suited for themodification depends on the particular protein (and its configuration).Where multiple putative phosphorylation sites (and phosphorylatablesites) are to be included in the modified protein, one would considerthe potential availability of either or both ends and other positions ofthe molecule for providing the amino acid recognition sequence. Thus, inaccordance with the invention, phosphorylation recognition sequences canbe introduced at any point in a naturally occurring protein sequenceproviding such introduced sequences do not adversely affect biologicalactivity where such activity is desired.

Once the recognition site for a particular protein kinase is identified,the invention provides a method for making by recombinant-DNA techniquesthe DNA sequence which encodes the recognition site for that kinasewithin, fused or linked to the DNA sequence encoding the functionalprotein which is to contain the corresponding putative labelling site.

The invention contemplates and includes any protein which isradio-labellable by the methods of this invention and which possesses atleast one of the properties of the corresponding unlabelled (orunlabellable) protein. In accordance with the invention, thenon-phosphorylated (or non-phosphorylatable) protein is modified tointroduce into the amino acid sequence the putative phosphorylatablesite; this is performed after having modified the DNA sequence encodingthe amino acid sequence of the protein with the DNA sequence (part orall) which codes for the putative phosphorylated site. In the case ofinterferons, the invention includes all interferons natural or“non-natural” interferons, including such structurally modifiedinterferon species which have been reported in the literature (such ashybrid interferons, modified interferons) as discussed above, and othermodified interferons which will be reported in the future.

Natural and “non-natural” (including modified) interferon species have avariety of biological activities and such activities are known to occurin different ratios; thus, the invention contemplates not onlyradio-labelled interferons which have any one of these properties (andin any ratio), but also biological or other properties not yetidentified in the known interferon species. It is recognized that thephosphorylation may modify one or more of the properties of the proteinto one degree or another (see 47, 100, 101, for example). Indeed thereare situations where the properties may be enhanced or developed wherethey were not detectable prior to modification of the protein.

The invention also provides particularly interesting labellable andlabelled proteins like phosphorylated antibodies (especially monoclonalantibodies, hybrid antibodies, chimeric antibodies or modifiedantibodies), hormones, and “modified” streptavidin. The modifiedstreptavidin can be bound to individual biotinylated antibodies, eachstreptavidin being modified by single or multiple phosphorylated groups,which product has greatly enhanced radiation and therefore diagnosticand therapeutic potential.

The invention also provides a hybrid interferon protein Hu-IFN-αA/gammaconstituted of Hu-IFN-αA to which there is fused the COOH-terminal 16amino acid region of Hu-IFN-gamma, which contains a putativephosphorylation site, and the hybrid interferon fusion protein labelledwith phosphorus. The fusion protein was synthesized with an expressionvector constructed by oligonucleotide-directed mutagenesis. Theinvention also provides the DNA coding sequence for the fused hybridinterferon protein, expression vectors and the transformedmicroorganisms, e.g. E. coli host and other suitable hosts describedbelow.

The foregoing is not intended to have identified all of the aspects orembodiments of the invention nor in any way to limit the invention. Theinvention is more fully described below.

The accompanying drawings, which are incorporated and constitute part ofthe specification, illustrate embodiments of the invention, and togetherwith the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures first illustrate the fused-hybrid interferon (FIGS. 1-7) andthen the other modified interferons made in accordance with theinvention (FIGS. 8-13).

FIG. 1 is a schematic illustration of a hybrid interferonHu-IFN-αA/gamma of Hu-IFN-αA and Hu-IFN-gamma, the Hu-IFN-gamma segmentrepresenting the 16 COOH-terminal amino acids and containing theputative phosphorylation site.

FIG. 2 shows an outline of the construction of the fusion protein fromHu-IFN-αA and Hu-IFN-gamma, the Hu-IFN-αA/gamma.

The EcoRI fragment from pRC23/IFN-gamma was ligated into the BstEII siteof pXZ-6 (pIFN-αA) by blunt-end ligation to yield pXZ-7. The EcoRIfragment of pXZ-7 was inserted into M13mp18 to yieldM13mp18/IFN-αA/IFN-gamma. Oligonucleotide-directed deletion wasperformed on this latter construct with the oligonucleotide shown toyield a recombinant containing the proper deletion(M13mp18/IFN-αA/gamma). The EcoRI fragment from this M13 recombinantreplaced the EcoRI insert of pXZ-6 to yield pXZ-8 (pIFN-αA/gamma), theexpression plasmid encoding the Hu-IFN-αA/gamma fusion protein. Theproper construction was confirmed by sequencing the recombinant. T4-trepresents the phage T4 transcription terminator (56, 57); Ap,ampicillin; cip, calf intestinal phosphatase.

FIG. 3 shows the nucleotide sequence encoding and amino acid sequence ofthe hybrid Hu-IFN-αA/gamma.

FIG. 4 shows a polyacrylamide gel electrophoresis of the purifiedproteins. Lane 1 represents Hu-IFN-αA; lane 2, the fused proteinHu-IFN-αA/gamma; and lane 3, Hu-IFN-gamma. The molecular weightstandards (STD) are shown in the right column. The gels were developedwith a silver stain.

FIG. 5 shows a polyacrylamide gel electrophoresis of phosphorylatedHu-IFN-gamma and the fusion protein Hu-IFN-αA/gamma. The left panel ofthe Figure represents the gel stained with Coomassie blue. The rightpanel represents the autoradiograph of the same gel as shown on the leftpanel. The heavy stained bands at MW_(R) 69,000 represent bovine serumalbumin which was added to the phosphorylation reactions.

FIG. 6 shows the binding of [³²P]Hu-IFN-αA/gamma to Daudi cells. Thebinding of [³²P]Hu-IFN-αA/gamma to Daudi cells was performed asdescribed under “Experimental Procedures”. Specific binding ()represents the difference between total binding (∘) and non-specificbinding (Δ) Non-specific binding represents the binding in the presenceof excess unlabelled Hu-IFN-α/A. The specific activity of[³²P]Hu-IFN-αA/gamma was about 141 μCi/μg.

FIG. 7 shows the binding of [³²P]Hu-IFN-αA/gamma to MDBK cells. Thesymbols and explanations are the same as in FIG. 6.

FIG. 8 shows nucleotide and amino acid sequences of the COOH terminus ofHu-IFN-αA, Hu-IFN-αA-P1, -P2, and -P3. The phosphorylation sitesrecognized by the cAMP-dependent protein kinase created in Hu-IFN-αA-P1,-P2, and -P3 are shown in rectangles. The sites contain the recognitionsequence Arg-Arg-Ala-Ser-Leu or Arg-Arg-Ala-Ser-Val (for phosphorylationof this Ser) for the cAMP-dependent bovine heart kinase together withadditional amino acids. The nucleotide sequences and amino acidsequences of the interferons are aligned for comparison.

FIG. 9 shows the construction of expression plasmids for Hu-IFN-αAcontaining phosphorylation sites.

FIG. 10 shows an analysis of Hu-IFN-αA-P proteins by SDS-polyacrylamidegel electrophoresis. The gel was stained with silver. Molecular weightstandards are shown on the left most lane.

FIG. 11 shows an autoradiograph of SDS-polyacrylamide gelelectrophoresis of the phosphorylated proteins. Hu-IFN-gamma serves asan internal molecular weight standard as in FIG. 10.

FIG. 12 shows the binding of [³²P]Hu-IFN-αA-P proteins to cells. Theinset represents Scatchard analysis of the specific binding data. “B”represents the radioactivity of ligands bound to cells and “F” is theradioactivity of free or unbound ligands. (A) and (B): binding[³²P]Hu-IFN-αA-P1 to Daudi and MDBK cells, respectively; (C) and (D):binding of [³²P]Hu-IFN-αA-P2 to Daudi and MDBK cells, respectively.

FIG. 13 shows the electrophoretic analysis of crosslinked[³²P]Hu-IFN-αA-P1, -P2 and -P3 receptor complexes.

The Figures are explained in greater detail hereinbelow.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In accordance with the invention, proteins which are normally notphosphorylatable can be modified to render them phosphorylatable. Inparticular, it was of great interest in conjunction with this inventionto determine whether and how one could achieve the phosphorylation of aparticular protein, the interferon Hu-IFN-αA, which as opposed toHU-IFN-gamma could not be phosphorylated (as discussed above). Themethodology to achieve this result (especially without loss of itsbiological activity) has provided the potential to modify other proteinsand render them phosphorylatable. Thus, in accordance with theinvention, HU-IFN-αA has been used as a model for phosphorylation ofproteins in general.

Hu-IFN-αA has been modified in accordance with the invention to yieldtwo embodiments, which will be described hereinafter. Both embodimentsare applicable to proteins in general and one skilled in the art willselect that most suitable under the particular circumstances.

The first embodiment is the construction of a phosphorylatable hybrid orfused protein, Hu-IFN-αA/gamma; the second embodiment is aphosphorylatable modified Hu-IFN-αA with an insert more fully describedbelow.

The following describes illustrative, but not limiting, specificembodiments.

Hu-IFN-αA/gamma, a Hybrid Protein of Hu-IFN-αA and the COOH-terminal 16Amino Acids of Hu-IFN-gamma

The following describes a hybrid or fused protein of Hu-IFN-αA andHu-IFN-gamma. The fused-hybrid protein was expressed by an expressionvector constructed by oligonucleotide-directed mutagenesis. Theconstruction of the expression vector, the expression and purificationof the protein, the phosphorylation of the functional hybrid protein andthe binding of [³²P]HU-IFN-αA/gamma to cells (MDBK) are described below.The hybrid protein has antiviral activity.

The fusion protein prepared in accordance with the invention comprisesHu-IFN-αA to which the COOH-terminal 16 amino acids of Hu-IFN-gamma wasfused. The protein was prepared by constructing an expression vector byoligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-αA/gammawas expressed under the control of the phage lambda P_(L) promoter. Theprotein was purified with a monoclonal antibody against Hu-IFN-α or theCOOH-terminal amino acid sequence of Hu-IFN-gamma. The purified proteinexhibited a single major band on sodium dodecyl sulfate-polyacrylamidegel electrophoresis and has antiviral activity on human and bovinecells. Unlike Hu-IFN-αA, but similar to Hu-IFN-gamma, it was found thatthe hybrid Hu-IFNαA/gamma can be phosphorylated by [gamma-³²P]ATP andcAMP-dependent protein kinase. The phosphorylated molecule binds to theIFN-αA/β receptor.

The introduction of a phosphorylation site into Hu-IFN-αA by fusion ofthe region of Hu-IFN-gamma which contains the putative phosphorylationsite provides a new reagent for studies of receptor binding,pharmacokinetics and other studies where labelled interferons areuseful.

Thus, in accordance with the invention, a hybrid protein was constructedby fusing the COOH-terminal end of Hu-IFN-gamma to the COOH terminus ofHu-IFN-αA to yield a hybrid protein which contains the putativephosphorylation site. The phosphorylated protein was found to bebiologically active.

The construction of a plasmid for expression of hybrid Hu-IFN-αA/gammawas carried out as follows.

As shown in FIG. 2, the DNA sequences for Hu-IFN-αA and Hu-IFN-gammawere cloned into M13mp18. Plasmid pXZ-8 encoding a fusion protein wasconstructed by oligonucleotide-directed deletion as shown.

Plasmid pXZ-8 is deposited with the ATCC under Accession number 40510,and designated as pHu-IFN-αA/gamma.

To screen for the plasmid recombinant with the proper deletion, colonyhybridization was performed on 200 colonies with the oligomer as probe(58) with the observation that 53 colonies were positive. Of the 53positive colonies, 36 were analyzed by restriction digestion with EcoRIwith the result that only two colonies contained the expectedrestriction fragments. DNA sequencing confirmed that the 5′ end of thesequence coding for the 16 COOH-terminal amino acids of Hu-IFN-gamma wasfused to the 3′ end of the coding sequence for Hu-IFN-αA (FIGS. 1, 2,and 3). A 732 bp fragment was deleted in the correct recombinant. Thegene for hybrid Hu-IFN-αA/gamma was constructed and expressed undercontrol of the lambda P_(L) promoter. The sequence of hybridHu-IFN-αA/gamma consists of 181 amino acids (FIG. 3).

As discussed further below, other promoters in various expressionvectors can also be used.

The characterization and phosphorylation of hybrid Hu-IFN-αA/gamma wasperformed as follows.

Hu-IFN-αA/gamma was induced when the E. coli RR1(pRK248cIts857) cells orE. coli AR68(pRK248cIts857) cells harboring the plasmid pXZ-8 were grownat 42° C. and the hybrid protein was purified by monoclonal antibodyaffinity chromatography as described hereinafter. The purified proteinexhibited a specific activity of 4×10⁸ units/mg. On SDS-polyacrylamidegel electrophoresis in the presence of 2-mercaptoethanol, it exhibited amajor single band which migrated slower than Hu-IFN-αA and almost at thesame position of Hu-IFN-αA-P3, another Hu-IFN-αA derivative with 182amino acids as shown in FIGS. 4 and 5. It was observed thatHu-IFN-αA/gamma is not as stable as Hu-IFN-αA. During experimentalprocedures, purification steps, and storage, degradation of the proteinextracted from E. coli RR1 was observed; much less degradation wasobserved when E. coli AR68 was used as the host cell.

It was found that hybrid Hu-IFN-αA/gamma can be phosphorylated with[gamma-³²P]ATP and bovine heart muscle cAMP-dependent protein kinase.The specific radioactivity of [³²P]Hu-IFN-αA/gamma was about 5 μCi/μgprotein in preparations isolated from E. coli RR1; in preparations ofHu-IFN-αA/gamma isolated from E. coli AR68, the specific radioactivitywas increased to about 141 μCi/μg, presumably reflecting greaterintegrity of the carboxyl terminus. These values are similar to therange of values obtained for [¹²⁵I]IFN-αA (59-62). [³²P]IFN-αA/gammamigrates as a single species at the same position as unlabelledIFN-αA/gamma on SDS-polyacrylamide gel electrophoresis in the presenceof 2-mercaptoethanol (FIG. 5).

The binding of [¹²]Hu-IFN-αA/gamma to cells was as follows.

Bovine MDBK cells were used for binding studies because Hu-IFN-αAexhibits high antiviral activity on this cell line and binds to it (16,28, 63), whereas Hu-IFN-gamma does not. To determine whetherHu-IFN-αA/gamma binds specifically to bovine MDBK and human Daudi cells,cells were incubated with various concentrations of [¹²P]Hu-IFN-αA/gammain the absence and presence of unlabelled Hu-IFN-αA. Specific binding of[³²P]Hu-IFN-αA/gamma to both KDBK and Daudi cells is shown in FIGS. 5and 7.

These results with [³²P]Hu-IFN-αA/gamma indicate that the fusion of thephosphorylation site of Hu-IFN-gamma to the CCCH terminus of Hu-IFN-αA,which itself cannot be phosphorylated, did not destroy the capability ofthe protein to bind. The Hu-IFN-αA/gamma protein also retained itsactivity.

This description of the first embodiment of the invention shows thesuccessful construction of a hybrid DNA sequence capable of coding foran amino acid sequence containing a putative phosphorylation site, theconstruction of a suitable expression venicle and of a transformed hostwhich readily expressed the hybrid protein with an intact putativephosphorylation site, and its phosphorylation.

The second principal embodiment of the invention reached the initialobjective in a somewhat different manner; yet it still resulted in amodified interferon which is phosphorylatable.

While in the first above-described embodiment, the DNA sequence codingfor the entire protein which is not phosphorylatable, in this caseHu-IFN-αA, is modified by fusion at the end of its sequence to asequence which codes for a putative phosphorylatable amino acidsequence, the second embodiment does not require the fusion to be at theend of the sequence and, in fact, does not require a fusion step.

Thus, the second embodiment shows the wider applicability of theinvention to proteins in general.

In the second embodiment the resulting protein has been modified byinsertion of an appropriate phosphorylatable sequence.

The second embodiment will be described hereinafter.

Hu-IFN-α Having Inserted Labellable Sites

An important concept of the invention is the incorporation of akinase-recognizable amino acid sequence into selected proteins (orconverting an amino acid sequence which is not recognizable by a proteinkinase into a sequence which is so recognized), and labelling theproteins which contain the putative kinase-recognizable phosphorylationsite by attaching a selected radioactive label by means of the catalyticaction of the kinase.

Generally, these steps will be performed at the level of the nucleotidesequence by DNA recombinant procedures. After the amino acid consensussequence recognizable by a protein kinase is identified, the nucleotidesequence encoding this amino acid sequence can be defined andconstructed. For purposes of this disclosure, a nucleotide sequenceencoding an amino acid sequence recognizable by a protein kinase will bedesignated a “PK nucleotide sequence”. Similarly, the amino acidsequence recognizable by a protein kinase will be designated “PK aminoacid sequence”. A PK nucleotide sequence can be introduced into anyselected nucleotide sequence, specifically a nucleotide sequenceencoding the amino acids of a protein, polypeptide, or peptide.Preferably, the PK sequence is inserted into or joined to a nucleotidesequence which codes for a protein in such a manner so that the proteinproduct is biologically, biochemically or otherwise active. The term“biologically active” is used throughout in this generic sense. Thus,the modified protein will be phosphorylatable and still be biologicallyactive. Additionally, the phosphorylated modified protein should also beappropriately active for the use desired. In some cases, thephosphorylated protein could be inactive biologically, yet still beuseful in a radioimmunoassay, for example.

It is only necessary in accordance with the invention that there beincorporated that much of the amino acid consensus sequence that willcontain or be the site for the phosphorylation. Likewise at the DNAlevel, it is only necessary that the codons be provided which code forthe amino acids which will be recognized by the kinase. Lack of absolutesubstrate specificity which has been reported to be a general propertyof protein kinases (21, 100), may provide further latitude in theselection of nucleotide sequences, or the codons coding for therecognized amino acids.

By “incorporates”, “incorporation” or “insertion” or equivalent term, itis not necessary that the amino acid recognition sequence (or thenucleotide sequence coding for that amino acid sequence (or partthereof) be actually within the desired protein (or the nucleotidesequence coding therefor); it is sufficient that it be part of themodified protein, or part of the nucleotide sequence (or of partthereof) coding for the modified product.

A specific embodiment of the concept of the invention is a geneticallyengineered Hu-IFN-αA of the invention which, unlike the known Hu-IFN-αA(native, natural or genetically engineered), is phosphorylatable and thephosphorylated modified Hu-IFN-α. The phosphorylated Hu-IFN-αA-P hasretained biological activity with high radio-specific activity(2,000-12,000 Ci/mmol). Phosphorylation was performed with the catalyticbovine heart kinase which is a cyclic AMP (cAMP)-dependent kinase. Theamino acid recognition (or consensus) sequences for the cAMP-dependentprotein kinase have been identified as Arg-Arg-Ala-Ser-Val andArg-Arg-Ala-Ser-Leu and Arg-Arg-Ala-Ser-Val-Ala amongst others (20, 21).In general cAMP-dependent protein kinases recognize the sequenceArg-Arg-Xxx-Ser-Xxx (21). The amino acid and nucleotide sequences ofHu-IFN-αA and their corresponding coding sequence have been reported(23-25).

Serine has been shown to be the target amino acid for phosphorylationfor both the murine (5) and human (4, 10) IFN-gamma. It has also beenshown that in Hu-IFN-gamma there are two serines capable ofphosphorylation by the cAMP-dependent protein kinase, serines 132 and142 (5, 10).

In accordance with the invention, in three specific illustrations ofphosphorylated Hu-IFN-α (herein designated as Hu-IFN-αA-P1, -P2, and-P3), the carboxyl-terminal amino acids were modified to contain thephosphorylation sites, and the nucleotide sequences encoding theputative phosphorylation sites were constructed.

The modified interferons were constructed with the use ofoligodeoxyribonucleotides designed to produce insertions andsubstitutions at the carboxyl terminus of Hu-IFN-αA by site-specificmutagenesis procedures (described in the literature and also furtherdescribed below) with the appropriate DNA sequences inserted into phageM13mp19. The general methods used for synthesizing a Hu-IFN-α with amodified carboxyl terminus in E. coli are further described in thefollowing references (15, 26, 27).

It is not necessary that the amino acids at the carboxyl terminus be theones that are modified; the modification can be at the amino terminalend of the sequence, and correspondingly can be towards the 5′ end ofthe nucleotide sequence; the modification can be virtually at anyposition of the protein sequence (and corresponding coding region) aslong as the phosphorylation site is recognizable by the kinase in theintact protein.

As illustrated above with respect to Hu-IFN-α, the introduction intonucleotide sequences of one or more putative sites encoding amino acidsequences recognizable by an enzyme like a protein kinase opens wide andimportant new possibilities for modifying proteins in which such sitesare absent, or inaccessible, or into which it is desired to introducesuch additional sites.

Amongst the protein kinases there are known cyclic nucleotide-dependentprotein kinases which catalyze the transfer of the gamma-phosphate ofATP to serine and/or threonine hydroxyl groups of acceptor proteinsubstrates, calcium-dependent protein kinases, tyrosine-specific proteinkinases, cyclic nucleotide- and calcium-independent protein kinases. Theinvention contemplates the use of any of the protein kinases, thenucleotide sequences which code for one or more of the correspondingputative sites for phosphorylation and the modified protein containingthe amino acid sequences recognizable by this selected protein kinase,and the phosphorylated protein containing the radio-label. Such labelcan be phosphorus, or sulfur, or other groups discussed herein. Also, itneed not be a radio-label. It is sufficient that it be an “identifiable”label.

Broadly considered, the invention contemplates the phosphorylation ofvarious proteins by protein kinases after the introduction ofphosphorylatable sites. Thus, the wide applicability of the invention inproviding appropriate sites for phosphorylation in proteins otherwisenot phosphorylatable can be recognized.

Throughout this description of the invention, the terms“phosphorylatable” (or “phosphorylated”) are used. Reference is alsobeing made to the analogs of the phosphate donor ATP, such as the gammathiophosphate analog. Through this description the term“phosphorylation” or like term is intended to be generic to include“thiophosphorylation” where for instance, ³¹P, ³²P, or ³²P is replacedby ³⁵S or sulfur analogs.

As discussed herein, kinases have been reported to catalyzephosphorylation not only at serine but also at threonine or tyrosine(21, 22, 64). Thus, it is within the scope of the invention to constructthe nucleotide sequence which codes for the putative site(s) ofrecognition for the kinase selected and, in a manner analogous to thatdescribed herein, construct the replicable expression vehicle, introduceit into an appropriate host which in turn will express the modifiedphosphorylatable protein. In appropriate cases, the modifiedphosphorylatable protein is exogenous and mature. The phosphorylatableproteins can then be phosphorylated of course likewise, analogs ofphosphorylated proteins—e.g., sulfur-labelled—can be made.

The nucleotide sequence encoding the putative phosphorylation site inthe case of modified Hu-IFN-α which is recognized by the cAMP-dependentprotein kinase is created by the oligodeoxyribonucleotide-directedinsertion on the level of DNA as shown in FIG. 9.

Construction of the Modified Interferons

The construction of expression plasmids for Hu-IFN-α containingphosphorylation sites was carried out as follows. The EcoRI-PstIfragment from pIFLrA that contained the coding sequence for Hu-IFN-αA(23, 24) was inserted into the EcoRI-PstI site of M13mp19 to formM13BL27 that was used as the template for site-specific alterations asshown in FIG. 9. To construct the coding sequences for Hu-IFN-αA-P1 andP2, two oligodeoxyribonucleotides were synthesized to anneal to M13BL27with the formation of a loop that permits the insertion of nucleotidesto generate a coding sequence for a phosphorylation site at theCOOH-terminal end (boxed residues, FIG. 8). The oligonucleotides used toprepare Hu-IFN-αA-P1 and -P2, respectively, were:AGT-TTA-AGA-AGT-AAG-AGA-AGG-GCA-AGT-GTT-GCA-TGA-AAA-CTG-CTT-CAA; andACA-AAC-TTG-CAA-AGA-AGT-TTA-AGA-AGG-GCA-AGT-TTA-GCA-TGA-AAA-CTG-CTT-CAA.The underlined nucleotides are homologous with the coding sequence and3′ non-coding nucleotide sequence of the cDNA encoding Hu-IFN-αA; thenucleotides not underlined produce a loop for the insertion ofadditional residues for P1 and P2. A site-specific mutation as well asan insertion was introduced with the P2 oligonucleotide. After annealingof the oligonucleotide to single-stranded DNA from M13BL27, the secondstrand was synthesized and then cut with restriction endonucleasesEcoRI-PstI. The EcoRI-PstI fragment obtained was then reinserted intothe EcoRI-PstI site of phage M13mp19 as shown in FIG. 9, and then E.coli were transformed with the duplex DNA. Incomplete duplexes (upperright of FIG. 9) do not yield the PstI-EcoRI duplex fragment. Thisexcision and religation step was introduced to increase the efficiencyof the site-specific mutations providing an overall yield of about 40%positive clones. RF DNA samples, prepared from individual phage M13plaques, were screened for the presence of the inserted EcoRI-PstIfragment. Positive clones (i.e., those with insertions; about 75-90% ofthe plaques) were sequenced by the known Sanger dideoxynucleotideprocedure (68) to identify the proper mutated recombinant and to confirmthe sequence. By this procedure, about 50% of the transformantssequenced contained the mutated coding sequence with the phosphorylationsite. The EcoRI-PstI fragments were then excised from the respective RFDNA preparations from the phages (M13BL28, M13BL29 and M13BL30) andreligated into the EcoRI-PstI site of pRC23t to yield the expressionvectors pBL281, pBL291 and pBL301 as shown in FIG. 9. The two EcoRI-PstIfragments originating from pRC23t were obtained by restrictionendonuclease digestion of an expression plasmid for Mu-IFN-β thatcontained the trp terminator just downstream from the IFN-β codingsequence.

During the construction of the Hu-IFN-αA-P1 expression vector, a clonewas isolated with a duplication of the -P1 oligonucleotide, a singlenucleotide deletion of one A of codon 164 for Lys, and a second deletionof 11 3′-terminal residues (AA-CTG-GTT-CAA) from the downstream -P1oligonucleotide. This series of steps generated an in-phase codingsequence for a new phosphorylation site on a slightly larger moleculedesignated Hu-IFN-αA-P3 (FIGS. 8 and 9).

The general recombinant DNA procedures employed herein have beendescribed elsewhere (15, 26, 27). These methods are incorporated hereinby reference. The use of vectors containing the phage lambda P_(L)promoter for cloning of recombinants and expression of proteins has beendescribed by a number of boratories (12, 48, 49, 70-77). A variety of E.coli strains lysogenic for wild type and mutant phage lambda orcontaining plasmids encoding the phage lambda repressor (12, 15, 48, 49,70-78) have been used for growth of plasmids and/or expression ofplasmid encoded proteins. For example, the following E. coli strainsamong others have been used for replication of plasmids and/or forexpression of proteins under control of the phage lambda P_(L) promoter:E. coli 294 (75; ATCC #31977), AR 13 (77), AR58 (77), AR68 (75), AR120(77, 74), C600 (49, 71; ATCC #33766), N99 (72; CC #33956), RR1 (66, 70;ATCC #31343), W3110 (77). With E. coli strains carrying atemperature-sensitive repressor (cI857), the cells can be grown at 30°C. to prepare plasmids; upon shifting the temperature to 42° C., therepressor is inactivated and the DNA sequence under control of the phagelambda P_(L) promoter expressed (49, 66, 70-73, 75). Thus, cells can begrown to a high density at 30° C., then induced to express the proteinof interest at 42° C. Alternatively, with the use of wild type phagelambda repressor (cI⁻), nalidixic acid can be used to induce expressionof genes under control of the phage lambda P_(L) promoter (77). In thework involving this invention, it has been found desirable to use thetemperature sensitive repressor (cIts857) to regulate expression of thegenes under control of the phage lambda P_(L) promoter. Although it wasnot found necessary to use protease deficient strains to achieve highproduction of recombinant proteins in E. coli (49, 70, 74, 77), strainslacking proteases (79, 80, 81) might prove beneficial in some cases (75,76).

Construction and identification of bacterial plasmids containing thenucleotide sequence for Hu-IFN-αA is described in references 23 and 24.The reports describe the isolation of recombinant plasmids carrying DNAsequences corresponding to Hu-IFN-α species. The E. coli strain K-12derivative RR1 can be considered a useful host in the present invention.

Site-specific mutagenesis which is well-suited for the present purposesis described in the following citations (15, 26, 27 and 43 at unit 8,supplement 2), which are incorporated herein by reference. Thebackground of the method is reviewed in reference 44. A description inthe patent literature is found in U.S. Pat. No. 4,751,077, which is alsoincorporated herein by reference.

In accordance with the invention, the nucleotide sequence coding for theamino acid consensus sequence (which will contain the putativephosphorylation site) may be inserted anywhere into the nucleotidesequence of Hu-IFN-αA. This can be observed from the oligonucleotidesequences shown in FIG. 8. However, the insertion of the nucleotidesequence is preferably made at a site in the nucleotide sequenceencoding Hu-IFN-αA so as to minimize an undesirable effect on thebiological activity of the resultant recombinant protein, when suchbiological activity is critical.

The Expression of Hu-IFN-αA-P by Bacteria

The expression of the modified interferons in E. coli transformed withthe expression plasmids encoding the Hu-IFN-αA-P proteins and thepurification of Hu-IFN-αA-P proteins were carried out as follows. Eachof the three above-named vectors was introduced into E. coli cellscontaining the plasmid pRK248cIts857. E. coli AR68 cells and E. coli RR1cells containing the compatible plasmid pRK248cIts857 harboring each ofthe pBL281, pBL291 and pBL301 plasmids expressed Hu-IFN-αA-P1, -P2, and-P3, respectively, under control of the P_(L) promoter and trpterminator at 42° C. The yields of Hu-IFN-αA-P1, -P2, and -P3 in E. coliAR68(pRK248) were higher than that in E. coli RR1(pRK248) harboring thesame plasmids. The products expressed in E. coli AR68 at 42° C. wereused to purify the Hu-IFN-αA-P1, -P2, and -P3 proteins by immunoaffinitychromatography as described further below. The data in Table 1 (below)show that greater than 50% of the antiviral activity was recovered.Hu-IFN-αA-P1, -P2, and -P3 were purified to a specific activity of1.2×10⁸, 1.1×10⁸ and 1.5×10⁸ units per mg protein, respectively.

Plasmid pBL281 is deposited with the ATCC under Accession number 40509,and designated as pHu-IFN-αA-P1.

TABLE 1 Purification of Hu-IFN-αA-P Proteins Antiviral activity SpecificHuman Total Yield Protein Activity Purification IFN-αA Step units (%) mgunits/mg factor αA-P1 1 1.6 × 10⁷ 100 32.6 4.9 × 10⁵ 1 2 1.8 × 10⁷ 11318.4 9.9 × 10⁵ 2 3 1.1 × 10⁷ 71 0.09 1.2 × 10⁸ 246 αA-P2 1 1.6 × 10⁷ 10034.3 4.7 × 10⁵ 1 2 1.2 × 10⁷ 75 12.9 9.3 × 10⁵ 2 3 0.9 × 10⁷ 56 0.08 1.1× 10⁸ 232 αA-P3 1 1.6 × 10⁷ 100 33.0 4.8 × 10⁵ 1 2 1.8 × 10⁷ 113 15.01.2 × 10⁶ 2.5 3 2.6 × 10⁷ 163 0.18 1.5 × 10⁸ 312

The characterization of Hu-IFN-αA-P and its ³²P-labelled product wascarried out as follows. The purified Hu-IFN-αA-P1, -P2, and -P3 proteinswere analyzed by SDS-polyacrylamide gel electrophoresis in the presenceof 2-mercaptoethanol (FIG. 10). A single band was observed on the silverstained gel with Hu-IFN-αA-P1 or -P2. Hu-IFN-αA-P2 migrated slightlyfaster than Hu-IFN-αA-P1 and a little slower than Hu-IFN-αA.Hu-IFN-αA-P3 yielded two bands, the slower migrating band being theintact molecule.

Hu-IFN-αA-P1, -P2, and -P3 were phosphorylated by the cAMP-dependentprotein kinase with [gamma-³²P]ATP to a specific radioactivity of2,000-12,000 Ci/mmol. Following phosphorylation, [³²P ]Hu-IFN-αA-P1 and-P2 migrate on SDS-polyacrylamide gel electrophoresis in the presence of2-mercaptoethanol as a single band with an apparent molecular weight of19,000-20,000 (FIG. 11) corresponding to the same positions as thesilver stained unlabelled band. The labelled Hu-IFN-αA-P3 migratesslower than the -P1 or -P2 products as expected. Since the fastermigrating P3 band is unlabelled (cf. FIGS. 10 and 11), it is likely thatthe COOH-terminal extension of -P3 that contains the phosphorylationsite is trimmed from the full length product to yield the fastermigrating form. Unlabelled Hu-IFN-αA and [¹²⁵I]Hu-IFN-αA were used asmolecular weight standards and controls (FIGS. 10 and 11).

Antiviral Activity of the New Interferons

The effect of phosphorylation on the antiviral activity of Hu-IFN-αA-P1,-P2, and -P3 was determined in a parallel experiment. Phosphorylation,it was found, has little or no effect on the antiviral activity of theHu-IFN-αA-P1, -P2, and -P3 measured with both bovine MDBK cells andhuman WISH cells.

Table 2, below shows the antiviral activity of non-phosphorylated andphosphorylated interferon alpha proteins.

TABLE 2 Effect of Phosphorylation on the Antiviral Activity ofPhosphorylated Human Interferon Alpha Proteins Human Antiviral ActivityIFN-αA [gamma-³²P]ATP MDBK WISH αA-P1 − 1.6 × 10⁶ 4.0 × 10⁶ + 1.6 × 10⁶2.4 × 10⁶ αA-P2 − 1.6 × 10⁶ 8.8 × 10⁶ + 0.9 × 10⁶ 8.8 × 10⁶ αA-P3 − 6.4× 10⁶ 4.8 × 10⁶ + 4.8 × 10⁶ 3.2 × 10⁶

Ability to Bind to Receptors

The ability of [³²P]Hu-IFN-αA-P to bind to receptors was shown asfollows. [³²P]Hu-IFN-αA-P1 and -P2 bind to bovine MDBK and human Daudicells (FIG. 12) with the specific binding approaching saturation athigher concentrations. Scatchard analysis of the data yielded thefollowing estimates. The bovine MDBK cells contain approximately 3,800and 9,450 receptors per cell calculated from the binding of[³²P]Hu-IFN-αA-P1 and -P2, respectively; Daudi cells, approximately1,650 and 4,900 receptors per cell. Dissociation constants (K_(d)) werecalculated to be 1.4×10⁻¹⁰ M for the binding of [³²P]Hu-IFN-αA-P1 toboth the human and bovine cells; and 3.5×10³¹ ¹⁰ M and 2.2×10³¹ ¹⁰ M forthe binding of [³²P]Hu-IFN-αA-P2 to human and bovine cells,respectively. Similar results are obtained with [³²P]Hu-IFN-αA-P3.

All these phosphorylated [³²P]Hu-IFN-αA-P derivatives bind to theHu-IFN-α/β receptor because their binding to Daudi cells wascompetitively blocked by Hu-IFN-αA and Hu-IFN-3, but not byHu-IFN-gamma.

Crosslinking of [³²P]Hu-IFN-αA-P to the Receptors

The crosslinking of [³²P]Hu-IFN-αA-P to the receptors was carried out asfollows. [³²P]Hu-IFN-αA-P1, -P2, and -P3 were each covalentlycrosslinked to the receptors by reaction with disuccinimidyl suberateafter binding to the cells (FIG. 13). The radioactive complexes migrateas several bands with molecular weights of 100K-200K from the Daudicells or as a broad band with an apparent molecular weight of about 150Kfrom the MDBK cells upon analysis by SDS-polyacrylamide gelelectrophoresis. The crosslinked complexes of the receptors on the cellswith [³²P]Hu-IFN-αA-P1, -P2, and -P3 appear to be the same onSDS-polyacrylamide gels, but differ from the crosslinked complexes of[³²P]Hu-IFN-gamma formed with human Daudi cells. Neither the crosslinkedcomplexes nor the free [³²P]Hu-IFN-αA-P1, -P2, and -P3 are seen ifexcess non-radioactive Hu-IFN-αA is included during the binding reaction(FIGS. 12 and 13).

Hu-IFN-β Containing A Phosphorylation Site

Likewise nucleotide sequences coding for a Hu-IFN-β-like protein, whichsequence contains a putative phosphorylation site recognizable by thecAMP-dependent protein kinase, is prepared.

The following procedure generates a modified Hu-IFN-β containing a siterecognizable by the cAMP.-dependent protein kinase from bovine heart.The PstI-BglII 363 bp fragment from the cDNA encoding Hu-IFN-β isexcised from the expression vector pFIFtrp69 (104). The 363 bp fragmentis inserted into the PstI and XmaI sites of phage M13mp18. First thePstI end of the PstI-BglII fragment is ligated to the dsDNA of M13mp18cut with restriction endonucleases PstI and XmaI. The BglII end of thefragment is then ligated to the M13mp18 vector with the use of aBglII-XmaI linker:

GATCTGCGCGCGC

ACGCGCGCGGGCC

which reconstructs BglII and XmaI sites. Since there is no BglII site inthe polylinker region of M13mp18, this BglII-XmaI linker is used topreserve the BglII site and permit cutting with XmaI for preparation ofone of the intermediate recombinants (see below and analogousconstruction in FIG. 8). The M13mp18 containing the 363 bp fragment fromthe 5′ end of the coding region of Hu-IFN-β formed M13-A that is used asa template for site specific mutation as follows. The site specificinsertion is made similarly to that described above for construction ofHu-IFN-αA-P1. To construct the coding sequence for Hu-IFN-β-P, themodified Hu-IFN-β, aoligodeoxyribonucleotide(CTT-ACA-GGT-TAC-CTC-CGA-AGG-GCA-AGT-GTT-GCA-TGA-AGA-TCT-GCG-CGC-GCC-CGG)is synthesized to anneal to M13-A with the formation of a loop thatwould permit the insertion of nucleotides to generate a coding sequencefor a phosphorylation site at the COOH-terminal end. The underlinedresidues of the oligonucleotide shown above are homologous with thenucleotides of the phage M13-A that contains the cDNA fragment encodingHu-IFN-β. A comparison of Hu-IFN-β and Hu-IFN-β-P is shown below:

160                165 Hu-IFN-β Leu-Thr-Gly-Tyr-Leu-Arg-Asn-ENDCTT-ACA-GGT-TAC-CTC-CGA-AAC-TGA160                165                170 Hu-IFN-β-PLeu-Thr-Gly-Tyr-Leu-Arg-Arg-Ala-Ser-Val-Ala-ENDCTT-ACA-GGT-TAC-CTC-CGA-AGG-GCA-AGT-GTT-GCA-TGA

The underlined residues of the modified Hu-IFN-β-P from 165-170represent the cAMP-dependent protein kinase recognition site. Thenucleotides of the oligonucleotide above that are not underlined producea loop for the insertion of residues 166-170 of Hu-IFN-β-P. Afterannealing of the oligonucleotide to single-stranded DNA from M13-A, thesecond strand is synthesized and then cut with restriction endonucleasesPstI and XmaI. The resultant PstI-XmaI fragment is then reinserted intothe PstI-XmaI site of phage M13mp18 as shown analogously in FIG. 8 andthen E. coli are transformed with the duplex DNA. This excision andreligation step is introduced to increase the efficiency of thesite-specific mutations. RF DNA preparations from individual phage M13plaques are screened for the presence of the inserted PstI-XmaIfragment. Positive clones (i.e. those with insertions) are sequenced bythe Sanger dideoxynucleotide procedure (68) to identify the propermutated recombinant and to confirm the sequence. By this procedure,several transformants sequenced contain the mutated coding sequence withthe phosphorylation site (M13-B). The PstI-BglII fragment is thenexcised from the RF DNA from the phage M13-B and religated into thePstI-BglII site of pFIFtrp69 to yield the expression vector similar tothe original vector. The general recombinant DNA procedures employedhave been described elsewhere (15, 26, 27, 43).

The phosphorylatable modified interferon is expressible as described(104), and can be purified following the procedure for the correspondingHu-IFN-β (65).

The following illustrative examples are not intended to limit theinvention in any manner whatsoever.

ILLUSTRATIVE EXAMPLES

Bacterial Strains, Enzymes and Chemicals. E. coli RR1 containing theplasmid pRK248cIts857 with the temperature-sensitive repressor of thephage lambda P_(L) promoter, was obtained from Robert Crowl (12). Theplasmid pPRK248cIts857 was introduced into E. coli AR68 (75) bytransformation.

Restriction endonucleases and polynucleotide kinase were from NewEngland BioLabs. The buffer conditions used were described by themanufacturer. The Klenow fragment of Escherichia coli DNA polymerase Iand T4 DNA ligase were from International Biotechnologies, Inc.; calfintestinal.phosphatase (c.i.p.) was from Boehringer-MannheimBiochemicals. Ligation reactions were carried out in the presence of lowmelting pointagarose gel (15, 66).

The catalytic subunit of cAMP-dependent protein kinase from the bovineheart muscle with a specific activity of 20,000 units/ml was obtainedfrom Sigma. [Gamma-³²P]ATP with specific radioactivity of 5,000 Ci/mmolwas obtained from Amersham; dithiothreitol (DTT), from Bethesda ResearchLaboratories; bovine serum albumin (BSA), from Miles Laboratories;acrylamide and N,N′-methylenebisacrylamide, from InternationalBiotechnologies, Inc. (IBI); sodium dodecylsulfate (SDS), from Sigma;and disuccinimidyl suberate (DSS), from Pierce Chemical Co.

Interferon and Protein Assays. Interferon activity was determined by acytopathic effect inhibition assay with vesicular stomatitis virus andbovine MDBK cells (13). All interferon titers are expressed in referenceunits/ml calibrated against the reference standard for human leukocyteinterferon (G-023-901-527) obtained from the Antiviral SubstancesProgram of the National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, Md. Protein concentration wasdetermined by the procedure of Bradford (14) with bovine serum albuminas a standard.

The following illustrates the preparation of the fused-hybridHu-IFN-αA/gamma.

Synthesis and Phosphorylation of Oligonucleotide

The oligodeoxyribonucleotide CTGACTCCTTTTTCGCTTT TCCTTACTTCTTAAC whichwas used for oligonucleotide-directed mutagenesis and hybridizationscreening was synthesized and phosphorylated as described (66). Afterthe phosphorylation reaction, the reaction mixture was diluted with 3.5ml of 6×SSC (0.9 M NaCl, 90 mM sodium citrate) and used directly forscreening by hybridization.

Oligonucleotide-directed Deletion. About 100 pmoles of phosphorylatedoligomer were annealed with 1 pmole of ssDNA template in 10 μl of 30 mMTris-HCl , pH 7.5, 10 mM MgCl₂ at 80° C. for 5 min. and gradually cooledto 30° C. for 30 min., then put on ice. The volume was adjusted to 20 μlof 30 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mMdGTP, 0.5 mM dTTP, 1 mM ATP, 10 mM dithiothreitol (DTT). The oligomerwas extended with 2.5 units of the Klenow fragment of DNA polymerase Iin the presence of 6 units of T4 DNA ligase at 16° C. for 24 hr. Thereaction mixture was then extracted once with an equal volume of phenoland chloroform (1:1) and precipitated with 1/10 volume of 2.5 M sodiumacetate and 2 volumes of 95% ethanol. After washing with 75% ethanol,DNAs were digested with SpeI for the enrichment of the mutated DNAbefore transformation of RR1(lambda-cIts857). For screening, the colonyhybridization method was used as described (58, 67). DNA sequencing wasperformed by the dideoxy sequencing method (68) after the 600 bpHincII/PstI fragment from M13mp18/Hu-IFN-αA/gamma was inserted into theHincII and PstI site of M13mp19.

Preparation and Purification of Hu-IFN-αA/gamma. E. coliRR1(pRK248cIts857) cells harboring Hu-IFN-αA/gamma were grown in LBbroth at 30° C. overnight. An overnight culture of 40 ml was diluted to2,000 ml of M9 medium containing 0.4% of casamino acids, 50 μg/mlampicillin (Ap) and 25 μg/ml tetracycline. Bacteria were grown at 30° C.until the cell density at 600 nm reached 1.0, at which time the cellswere transferred to 42° C. for 2-3 hrs. The cells were collected bycentrifugation at 7,000 rpm for 10 min. and stored at −20° C. untilused.

For purification, all steps were carried out at 4° C. Frozen cells (10g) were suspended in 3 volumes (40 ml) of 7 M guanidine hydrochloride in25 mM Tris-HCl, pH 7.5. The suspension was mixed for 1 hr. andcentrifuged at 17,000 rpm for 30 min. The supernatant was diluted into10 volumes of phosphate buffered saline (PBS). Solid ammonium sulfatewas added to 65% saturation with vigorous stirring. The suspension waskept at 4° C. for 2 hrs. and then centrifuged at 10,000 rpm for 20 min.The pellet was suspended in 20 ml of PBS and dialyzed extensivelyagainst PBS. The suspension obtained was centrifuged at 10,000 rpm for20 min., and the supernatant was mixed with 0.3 ml of monoclonalantibody to Hu-IFN-αA (LI-8) coupled to Affi-gel 10 (16, 41) for 1 hr.The mixture was then loaded into a 1-ml disposable syringe. Afterwashing with four column volumes (0.3 ml) of 0.5 M NaCl, 25 mM Tris-HCl,pH 7.5, the column was rinsed with four column volumes of 0.15 M NaCland then eluted with four column volumes of 0.2 N acetic acid and 0.15 MNaCl, pH 2.5. Antiviral activity was eluted in the first two fractions.It was measured by a cytopathic effect inhibition assay on bovine MDBKcells (13). The concentration of protein was determined by the procedureof Lowry (69) or Bradford (14) with bovine serum albumin as a standard.The concentrations obtained by the two procedures were in agreement. Theprocedure yielded 240 μg of Hu-IFN-αA/gamma with a specific activity of4×10⁸ units/mg.

Preparation of Hu-IFN-αA/gamma protein from E. coli AR68(pRK248cIts857)harboring the expression plasmid pXZ-8 and its purification byimmunoaffinity chromatography with monoclonal antibody against Hu-IFN-αis as described below for the Hu-IFN-α-P proteins. The yield from 50 mlof bacterial culture was 388 μg of Hu-IFN-αA/gamma with a specificactivity of 2.3×10⁷ units/mg. The procedure of purification byimmunoaffinity chromatography with monoclonal antibody against theCOOH-terminal sequence of Hu-IFN-gamma is the same as above. Elutionwith acid yielded 281 μg of Hu-IFN-αA/gamma with a specific activity of1.3×10⁷ units/mg from 50 ml of bacterial culture.

Phosphorylation of Hu-IFN-αA/gamma. Hu-IFN-αA/gamma was phosphorylatedas described for Hu-IFN-gamma (3, 6) with some minor modifications.About 1 μg of Hu-IFN-αA/gamma was incubated at 30° C. for 60 min. with0.5 mCi of [gamma-³²P]ATP (>5,000 Ci/mmol, Amersham Corp.) and 10 unitsof the catalytic subunit of bovine heart cAMP-dependent protein kinase(Sigma) in 30 μl of 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 12 mM MgCl₂,and 3 mM DTT, then cooled on ice to stop the reaction. After addition of0.3 ml of 5 mg/ml bovine serum albumin in 10 mM sodium pyrophosphate, pH6.7, the reaction mixture was dialyzed extensively against 10 mM sodiumpyrophosphate, pH 6.7, at 4° C. Incorporation of radioactivity intoHu-IFN-αA/gamma was measured with a liquid scintillation spectrometerafter precipitation of the protein with trichloroacetic acid (82).

Binding of Hu-IFN-αA/gamma to cells. Bovine MDBK cells were used forbinding studies. MDBK cells were grown to confluence in 6-well tissueculture plates in medium (Gibco F-11) supplemented with 10% fetal calfserum and 50 μg/ml gentamicin. Medium was removed, and 1 ml of freshmedium was added into each well. After 20 min., [³²P]Hu-IFN-αA/gamma wasadded in the absence or presence of 0.6 μg unlabelled Hu-IFN-αA (10⁸units/mg). Following incubation at room temperature for 1 hr., theplates were put on ice and each well was washed with three 1 ml volumesof cold PBS. Then 1 ml of 1% SDS was added to remove cells from thewells. Radioactivity was determined with a liquid scintillation counterby placing the 1 ml samples in 10 ml of a scintillation fluor (83).Binding of [³²P]Hu-IFN-αA/gamma to the human Daudi cells is describedbelow with the Hu-IFN-αA-P proteins.

The following illustrates the construction of modified interferons by“insertion”.

Construction of Modified Interferons. Because of certain limitations ofthe hybrid-fusion procedure and product as discussed above, analternative construction was explored. It was then discovered that aputative phosphorylation site could be introduced into a nucleotidesequence of Hu-IFN-α.

The amino acid recognition or consensus sequences for the cAMP-dependentprotein kinase have been identified as Arg-Arg-Ala-Ser-Val andArg-Arg-Ala-Ser-Leu among others (20, 21). The amino acid sequence ofHu-IFN-αA and its corresponding coding sequence have been reported(23-25). Hu-IFN-αA as well as other interferons have been expressed inE. coli expression vectors under control of the trp and the phage lambdaP_(L) promoter (see references 1, 24 and 25 for reviews and additionalcitations). The terminal nucleotides corresponding to the last tencarboxyl-terminal amino acids of Hu-IFN-αA are shown in FIG. 8 as wellas sequences corresponding to modified molecules, Hu-IFN-αA-P1, -P2, and-P3, which contain putative phosphorylation sites. To construct thesemolecules oligodeoxyribonucleotides were synthesized to introduce theinsertions and substitutions shown at the carboxyl terminus of Hu-IFN-αA(FIG. 8) by site-specific mutagenesis procedures with the appropriateDNA sequences inserted into phage M13mp19 (15, 26, 27). Thephosphorylation sites in Hu-IFN-αA-P1, -P2, and -P3 (FIG. 8) recognizedby the cAMP-dependent protein kinase were created by theoligodeoxyribonucleotide-directed insertion on the level of DNA as shownin FIG. 8.

The expression plasmids pBL281, pBL291 and pBL301, coding forHu-IFN-αA-P1, -P2, and -P3, respectively, were constructed as outlinedin FIG. 8. The sequences coding for the modified interferons wereinserted into an expression vector under control of the phage lambdaP_(L) promoter also as illustrated in FIG. 8. E. coli AR68 containingthe compatible plasmid pRK248cIts857 was transformed with each of theexpression plasmids encoding the Hu-IFN-αA-P proteins containing theCOOH-terminal sequences shown in FIG. 8. The phosphorylatable modifiedinterferons were expressed and purified as described further below.

Expression and Preparation of Hu-IFN-αA-P. E. coli RR1 (pRK248cIts857)cells harboring pBL281, pBL291 or pBL301 plasmids containing theHu-IFN-αA-P1, -P2, and -P3 coding sequences were grown at 30° C.overnight in M9CA medium (15) with the concentration of some componentsmodified as follows: 1% casamino acids, 1% glucose, 10 mM MgSO₂ and 1 mMCaCl₂, and containing 2 μg/ml thiamine, 50 μg/ml ampicillin and 12.5μg/ml tetracycline. For expression of Hu-IFN-αA and modlfled proteins,100 ml of M9CA medium was inoculated with 3-5 ml or an overnigntculture. The bacteria were grown at 30° C. until the cell densityreached an optical density a 600 nm of 0.3-0.5 in 2-3 hours, at whichtime the culture was transferred to 42° C. for an additional two hours.The bacterial cells were collected by centrifugation and lysed in 8 Mguanidine hydrochloride and 50 mM Tris-HCl, pH 7.6, at 0° C. for 10 min.The supernatant obtained after centrifugation at 14,000 rpm (SA-600Sorvall rotor) for 30 min. was used to assay the antiviral activity orto purifzey the Hu-IFN-αA and modified interferons.

E. coli AR68(pRK248cIts857) cells harboring plasmids pBL28, pBL291, orpBL301 containing Hu-IFN-α-P coding sequences were grown in LB medium(15) containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline at 32°C. overnight. The overnight culture was diluted five-fold with fresh LBmedium containing the same concentration of antibiotics as above andthen grown at 32° C. for 2-3 hours For expression of the Hu-IFN-αA-Pproteins, harvesting of cells, and preparation of supernatants theprocedures described above for use with E. coli RR1 cells were used.

The purification of Hu-IFN-αA proteins were carried out as follows. Allsteps for purification of Hu-IFN-αA-P species were carried out at 4°C.-8° C. Ten ml of the guanidine hydrochloride supernatant from 100 mlof expressed culture were diluted ten-fold with cold phosphate-bufferedsaline (PBS) and precipitated at 65% saturation of ammonium sulfate at4° C. overnight. The precipitate was collected by centrifugation at10,000 rpm (Sorvall GSA rotor) for 20 min. at 5° C. The supernatant wasdecanted and saved. The residual pellet was dissolved again in 10 ml ofcold PBS and the solution centrifuged as above. The combinedsupernatants of 30 ml were mixed with 1 ml of Affi-gel 10 to whichmonoclonal antibody LI-8 (against Hu-IFN-α) was Linked (16, 41) and thesuspension rocked at 4° C. for 1 hour. The immunoabsorbent was loadedinto the barrel of a 2 ml disposable syringe and washed with 20 columnvolumes or more of each of the following cold solutions sequentially(16): PBS, Buffer F (0.5 m NaCl 25 mM Tris-HCl, pH 7.5, and 0.2% TritonX-100); and 0.15 M NaCl. Then the interferon was eluted with Buffer H(0.2 M acetic acid, 0.15 M NaCl, pH 2.6) and 0.4 ml fractions collected.The eluted fractions were neutralized with 1 M Tris base to pH 7.0 andthe fractions of peak antiviral activity pooled.

The phosphorylation of Hu-IFN-gamma and Hu-IFN-αA-P proteins was carriedout as follows: Hu-IFN-αA-P or Hu-IFN-gamma were labelled with[gamma-³²P]ATP and the cAMP-dependent protein kinase as described forHu-IFN-gamma with some minor modifications (3, 6). About 0.25 to 0.65 μgof Hu-IFN-gamma or Hu-IFN-αA-P was incubated at 30° C. for 1 hour with0.25 mCi of [gamma-³²P]ATP (5,000 Ci/mmol, Amersham) and 7.5 units ofthe catalytic subunit of cAMP-dependent protein kinase in 30 μlcontaining components as previously reported (3, 6). The reactionmixture was then cooled in an ice bath, and, after the addition of 270μl of 5 mg/ml bovine serum albumin in 10 mM sodium pyrophosphate(NaPPi), pH 6.7, was dialyzed extensively against 10 mM NaPPi at 4° C.The radioactivity associated with [³²P]Hu-IFN-αA-P was determined in aBeckman Model-LS3801 scintillation spectrometer. The phosphorylatedHu-IFN-αA-P was stored in liquid nitrogen in small volumes.

It has been reported (6) that the Hu-IFN-gamma phosphorylated with ³²Phas a 100-fold higher specific radioactivity than reported for[¹²⁵I]IFN-gamma.

The phosphorylated interferons in accordance with the invention providesmolecules with higher radio-specific activity than previously obtainable(1,000-12,000 Ci/mmol) with retention of biological activity. Thus, thephosphorylation site inserted into Hu-IFN-αA at the COOH terminus doesnot detrimentally affect the biological activity (antiviral activity)and can be effectively recognized by the cAMP-dependent protein kinase.

Further Hu-IFN-αA-P1 and -P2 are stable during purification andphosphorylation. The Hu-IFN-αA-P3, which as shown in FIG. 8 has anadditional septidecylpeptide at the COOH terminus, degraded into atleast two fragments.

It is evident that for some biological applications the phosphorylatedmodified interferons, and those labelled with phosphate analogs such asthose containing S, should be stable in serum.

The binding of [³²P]Hu-IFN-αA-P1, -P2, and -P3 to bovine MDBK cells andhuman Daudi cells was performed. [³²P]Hu-IFN-αA-P1, -P2, and -P3crosslinked to cells exhibited one complex of about 150 K with thebovine MDBK cells and several complexes of 100-200 K with the humanDaudi cells.

The binding of [³²P]Hu-IFN-αA-P to cells was performed as follows:Confluent monolayers of bovine SBBK cells were trypsinized and 1 ml(1×10⁶ cells) of the cell suspension in Dulbecco's modified Eagle'smedium (Gibco) containing 10% inactivated fetal calf serum and 1%penicillin-streptomycin solution (Gibco) was added to each well of a6-well plate. The cell monolayers reached confluence and approximatelydoubled on overnight incubation at 37° C. at which time they were usedto measure binding of [³²P]Hu-IFN-αA-P. For beginning the binding, themedium was removed, then 1 ml of fresh medium containing[³²P]Hu-IFN-αA-P at the indicated concentration was added into each wellin the absence (−) or presence (+) of excess nonradioactive Hu-IFN-αA asa competitor (>500-fold more than [³²P]Hu-IFN-αA-P added). The plateswere incubated with rocking at room temperature (24° C.) for 60 min.,after which they were placed on ice to cool. Each well was washed threetimes with 1 ml of cold PBS to remove the unbound radioactive ligand.After washing, 1.5 ml of 1% sodium dodecylsulfate in water was added toeach well and, after dissolution of the cells and bound radioactivity,the entire 1.5 ml was counted in a Beckman Model LS3801 scintillationcounter in 2 ml Hydrofluor scintillation fluid.

The binding of [³²P]Hu-IFN-αA-P to human Daudi cells was performed asdescribed previously (17) with some modifications. Daudi cells wereharvested by centrifugation at 1,000×g for 10 min., washed twice withthe growth medium (RPMI-1640, Gibco-H18, supplemented with 12.5 mMsodium HEPES, 10% fetal calf serum and 50 μg/ml gentamicin) andresuspended in the medium to a concentration of 1×10⁷ cells/ml. Thebinding of [³²P]Hu-IFN-αA-P at the indicated concentration to 1.25×10⁶Daudi cells in a total volume of 125 μl was allowed to proceed in theabsence or presence of non-radioactive Hu-IFN-αA as a competitor at roomtemperature (24° C.) for 60 min., with gentle resuspension every 15 min.At the end of the 60 min. incubation period, 100 μl of the cellsuspension was layered onto a 300 μl cushion of 10% sucrose in PBS in asample cup and pelleted by centrifugation (Beckman Microfuge Type B) for2 min. Tubes were frozen in a dry ice-ethanol bath, and then the tips oftubes containing the cell pellets were cut off and counted as above. Thespecific binding at a given concentration of [³²P]Hu-IFN-αA-P is definedas the difference in bound radioactivity between samples incubated inthe absence (total) and presence (nonspecific) of excess non-radioactiveHu-IFN-αA.

The covalent crosslinking of [³²P]Hu-IFN-αA-P to the receptors wascarried out as follows: A monolayer of bovine MDBK cells in 75-cm²tissue culture flasks was washed twice with Dulbecco'sphosphate-buffered saline, trypsinized with 2 ml of trypsin-EDTAsolution (1× in phosphate-buffered saline, Gibco laboratories) at 37° C.until the cells were released from the tissue culture flask. Afteraddition of 10-20 ml of Dulbecco's modified Eagle's medium (GibcoLaboratories) containing 10% inactivated fetal calf serum and 1%penicillin-streptomycin (Gibco), cells were collected by centrifugationat 500×g for 5-10 min. and resuspended in the same medium to aconcentration of about 1×10⁷ cells/ml. About 5×10⁶ cpm of[³²P]Hu-IFN-αA-P (2,000-12,000 Ci/mmol) was added to 0.5 ml of cellswith or without 1 μg of non-radioactive Hu-IFN-αA as a competitor. Afterincubation with rocking at room temperature (24° C.) for 1 hour, thecells were pelleted for 20 seconds at 14,000 rpm in an EppendorfMicrofuge, washed twice with 1 ml of cold PBS, then treated with a finalconcentration of 0.5 mM disuccinimidyl suberate (freshly prepared indimethylsulfoxide) at 4° C. for 20 min. as described (7). Thecrosslinking of [³²P]Hu-IFN-αA-P to cell receptor proteins was analyzedby sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (18)on 1.5-mm thick slab gels containing 8% acrylamide as described indetail previously (6, 7). The crosslinking thus was confirmed.

³⁵S-labelled proteins, like Hu-IFN-αA-P1, are obtainable in a mannersimilar to that described above.

SDS-polyacrylamide Gel Electrophoresis. The proteins, labelled productsand covalent crosslinked complexes were analyzed by electrophoresis onthe SDS-polyacrylamide slab gels of 1.0 or 1.5-mm thickness by themethod of Laemmli (18). After electrophoresis, the proteins were stainedwith silver (19) or with Coomassie brilliant blue. Gels containingradioactive samples were dried under vacuum and autoradiographed at roomtemperature for the labelled ligands and at −170° C. for the covalentcrosslinked complexes with Kodak X-Omat film and intensifying screens.

Other Expression Vectors, Host Cells, Etc. While presently preferredprocedures to express the modified interferons, to make variousnucleotide sequences, and to transform specific hosts have beenillustrated, it is evident that the invention is not in any way limitedby these illustrations. Both eukaryotic and prokaryotic host cells maybe used. Several procedures for the isolation of genes and expression ofinterferons in bacterial cells and heterologous cells are quitewell-suited for production of modified interferons of the invention. Forinstance, several such methods describe in reference (1) Section VII theuse of yeast vectors for production (Chapter 59) of and secretion(Chapter 60) of human interferons by yeast. Other microbial strains ofE. coli may be used, or Bacilli, like Bacillus subtilis, Salmonellatyphimurium (as disclosed in U.S. Pat. No. 4,727,138) with plasmids thatcan replicate and express heterologous gene sequences therein. Otherexpression vectors are illustrated in U.S. Pat. No. 4,559,300, forinstance. Numerous other promoter systems (than the one illustratedherein) can be used like the trp (104, 105) and lac (104, 106) promotersfor example. All such references are Included hereby by reference.

Likewise, the modified interferons can be produced from vertebrate cellcultures, for instance, a COS-7 line of monkey kidney fibroblasts can beused as the host for the production of the modified interferons withappropriate expression vectors (see Chapter 63 in reference 1, forexample); other cell lines are suitable and are known. An example of theuse of a retroviral based vector for expression in eukaryotic cells isgiven in Chapter 56 of reference 1. Many other examples of eukaryoticexpression vectors have been described (see for example 50, 51, 84-90).

Vectors useful in the invention to replicate in a transformed host cellhave a DNA segment containing a functional origin of replication(replicon). Plasmids and phage DNA by their very nature containreplicons facilitating replication in a host cell. The vector will havea DNA segment which conveys to a transformable host cell a propertyuseful for selection of transformed cells from nontransformed cells. Anyof a wide range of properties can be used for selection purposes. One ofthe most commonly used properties is antibiotic resistance, e.g.,tetracycline resistance or ampicillin resistance.

The foregoing two elements generally are present in readily availableand recognized cloning vectors. Examples of suitable cloning vectors arebacterial plasmids, such as plasmids from E. coli, including pBR322,pMB89, ColEl, pCRl; wider host range plasmids, including RP4; phageDNAs, such as lambda, and the like. Most, if not all, of theabove-recognized vectors already carry the aforedescribed two elements.Of course, as is known, in certain recombinants the DNA need not containa replicon nor an attached marker.

Thus, any suitable expression vector may be used to express the modifiedinterferons having putative phosphorylation sites in accordance with theinvention.

In accordance with known procedures, the DNA comprises the regulatingsegments and the coding region. Thus, it is evident that the inventionis not limited to the particular means of constructing geneticrecombinants disclosed as illustrations and that one of average skill inthe art would without undue experimentation adapt, change or select theprocedures best suited to his objective.

For techniques and additional materials (vectors, host systems,plasmids, enzymes used in molecular cloning, synthesis and cloning ofcDNA, introduction of plasmid and bacteriophage lambda DNA into E. coli,etc.), see (15) and (43), incorporated herein by reference.

ADDITIONAL GENERAL OBSERVATIONS

As has been described above, Hu-IFN-αA and Hu-IFN-β cannot bephosphorylated (by the cMIP-dependent bovine heart kinase) whereas ithas been shown that Hu-IFN-gamma and the corresponding Ra- andMu-IFN-gamma are amenable to phosphorylation without modification of theDNA-sequence (5, 11).

Thus, in accordance with the invention, additional phosphorylation sitescan be introduced into Hu-IFN-gamma to provide interferon proteins thatcan be labelled to higher radio-specific activities than proteins, e.g.,interferons, with only a single phosphorylation site.

Thus, it is within the contemplation of the invention to increase thenumber of sites (phosphorylation, thiophosphorylation) in proteins whichalready have one (or a larger number) of such sites.

The origin of the phosphorylatable nucleotide sequence can furthermorebe varied. Instead of using the PK nucleotide sequence corresponding toHu-IFN-gamma, there may be used a PK sequence corresponding to any otherinterferon (such as murine interferon), or for that matter the PKnucleotide sequence is derivable from any other nucleotide sequenceencoding a protein known to be phosphorylatable. In this manner highlyinteresting phosphorylatable proteins (and phosphorylated proteins) canbe made available for practical applications.

Furthermore, it is within the scope of the invention to radio-labelproteins, like Hu-IFN-αA and -β which have not been phosphorylatableheretofore with a radioactive label which has advantages over iodinelabelling. Accordingly, it is within the scope of the invention to usethe sulfur analogs of the radioactive ATP wherein sulfur is substitutedfor the phosphorus. For instance, the gamma ³⁵S analog of ATP could beincorporated into the protein at the appropriate recognition site. Thus,the invention contemplates the use of the isotopes of sulfur andphosphorus, like ³⁵S , ³⁸S ³¹P ³²P, and ³³P. Some of these isotopes havenot yet been widely used because they are less readily available orbecause of their respective half-life. Lists of isotopes are of courseavailable. Any isotope which can be introduced into a compound to belabelled is contemplated by this invention.

From Table 3 shown below, it can be observed that ³⁸S with a half-lifeof 2.87 hours and an energy of 1.1 Mev may well be an ideal source forirradiation of tumors and other tissues when radiation is deemedappropriate. Proteins phosphorylated with such an isotope with a shorthalf-life of 2.9 hours would have a specific activity about one hundredtimes the specific activity that is obtainable with ³²P. The shorthalf-life of the isotope also has the advantage that it is eliminatedfrom the patient within a few days. Furthermore, the ability to generatespecific activities one-hundred fold that of ³²P permits the use ofsmaller dosages of the proteins so that antigenic side-effects (that is,antibody production to these proteins) is minimized, The ³³P phosphateproduces a β particle with an energy approximately one-seventh that of³²P. The isotope has a half-life of 24.2 days so that a proteinphosphorylated with a phosphate analog containing ³³P would haveapproximately one-half the specific activity of the analog labelled with³²P (with a 14.2 day half-life). The ³³P radiation would affect less ofthe area surrounding the tumor cells than the derivatives containing³²P. Thus, the use of the products of the invention can bring aboutimportant beneficial advantages.

ADDITIONAL APPLICATIONS AND USES OF MODIFIED PROTEINS

The interferons modified in accordance with the invention by thepresence of one or more phosphorylated groups—or analogs thereof likesulfur—and proteins so modified, have numerous applications and uses inthe biological, medical, biomedical (including therapeutic anddiagnostic), and other sciences.

It is contemplated that modified proteins in accordance with theinvention can have additional specific uses. A few illustrations of suchuses are described below.

1. Pharmacokinetics of Proteins.

It is often useful to follow the fate of injected proteins in animalsand patients. It is shown below that the phosphorus attached to some ofthese proteins is relatively stable in human and fetal calf serum; thusthe pharmacokinetics of proteins can be conveniently studied. Thus,phosphorylated proteins are especially well-suited for suchapplications.

For uses of the phosphorylated proteins or analogs of the inventionwhere the protein is expected to be in contact with human or animalserum, it is necessary that the protein derivative be stable in human oranimal serum. The derivative protein should be stable in the serum ofthe species in which the pharmacokinetic studies (or application) are tobe carried out, or in a serum equivalent, i.e., from the biologicalpoint of view, to the serum of the species on which the work is to beperformed.

For instance, in the work described above, the phosphate linked toHu-IFN-αA-P1, -P2, and -P3 is stable in fetal calf serum at 37° C. Inthe presence of human serum, the phosphate linked to Hu-IFN-αA-P1 and toHu-IFN-αA-P3 was stable at 37° C., but the phosphate linked toHu-IFN-αA-P2 was labile. After 6 and 16 hours at 37° C. approximately62% and 74% of the phosphate was hydrolyzed from [³²P]Hu-IFN-αA-P2.Thus, for applications where the stability of the phosphorylatedderivative is critical, a serum-stable derivative will be used. Similarconsiderations apply to modified Hu-IFN-β. For applications wherestability in serum is not essential, the serum-unstablephosphorylated—or analog—may be used.

The applications described herein are not limited to proteinsphosphorylated at the serine residue; it has been described above howkinases phosphorylate other amino acids such as threonine or tyrosine(20, 21, 23). Thus, proteins modified at these amino acids are withinthe contemplation of the invention. Because of the configuration of suchderivatized labelled proteins, it is not to be excluded that theirstability in serum may be improved if the correspondingserine-phosphorylated derivative is not adequately serum-stable.

2. General Diagnostic Reagents.

Additional specific applications of the modified proteins of theinvention are noteworthy. As referred to herein, virtually all proteinscan be engineered to introduce single or multiple phosphorylation (oranalog) sites. Such proteins can be used for a wide variety ofscientific purposes: to study the fate of these proteins in animals orhumans; to study their stabilities; or for use as any laboratory reagentwhere a radioactive protein is useful.

For example, molecular weight standards are commonly used forpolyacrylamide gel electrophoresis. Proteins with phosphorylation siteswould make convenient autoradiographic markers such as molecular weightmarkers, isolectric focusing markers or other markers. For suchapplications the serum stability is generally not critical, nor is theretention of the biological activity of the protein, e.g., theinterferon. Thus, for certain uses or applications it is not essentialthat a phosphorylatable protein in accordance with the invention havebiological activity.

3. Anticancer Therapeutic “Bomb”.

A particularly noteworthy and interesting application made possible bythe invention is what has been called here in the vernacular, atherapeutic or more specifically an antitumor “therapeutic radiationbomb”. Such a biologically-active composition uses biotin coupled to atumor-specific monoclonal antibody (Mab) (or to Fab or Fab′ fragments ifmore appropriate), and a multiple “modified” streptavidin bound to eachMab-bound biotin, each streptavidin being modified in that it hasmultiple phosphorylated groups. Since streptavidin is itself a tetramer,multiple radioactive groups are thus provided. These multipleradioactive groups expose the tumor with radiation which is greatlyamplified and hence more readily detectable and would produce greatertumor destruction. In the case where it is highly phosphorylatable it ismuch more easily detectable. Thus, each one of the biotins which isbound to each tumor-specific Mab binds tightly to the multiplestreptavidin molecules which in turn contain multiple labelledphosphorus atoms, or their equivalent isotopes.

It is evident that depending on the therapeutic or diagnosticobjectives, all streptavidins may be radioactive-phosphorus labelled orpartially or totally radioactive-thiophosphorus labelled, or labelledwith different phosphorus or sulfur isotopes, which have different decaymodes or levels of radiation energy. Such isotopes are discussed below.

Because antibody molecules are themselves multichain molecules, manysites can be introduced into the antibodies or Fab fragments directly bythe procedures of this invention.

4. Hormones, Cytokines, Lymaiokines, Growth Factors.

Hormones labelled with radioactive phosphorus or sulfur are anotherclass of biological materials within the scope of this invention. Forinstance, phosphorylated (e.g., ³³P, ³²P) hormones can be bound tospecific cell types differentially over other tissues. Cancerous tissuescontaining increased number of receptors for such hormones can betreated with appropriately phosphorylated hormones which will thenspecifically bind to these cells; thus therapy will be significantlyimproved.

Further, labelled hormones are commonly used for receptor studies toexamine their binding to cell surface receptors, to soluble receptors orother reagents and materials.

Typical of the labelled hormones (³³P, ³²P) contemplated by theinvention are growth hormone, insulin, FSH; LH, and others. It isevident such hormones genetically constructed lend themselves to theintroduction of one or more putative phosphorylatable orthiophosphorylatabie groups.

As noted above for hormones, the same considerations apply to cytokines,lymphokines, growth factors (i.e., IL-1, IL-2, IL-3, TNF-α, TNF-β, thevarious CSF molecules, erythropoietin EGF, NGF and others) and anyproteins with cell and/or tissue specificity to one degree or another.

5. Antibodies.

Streptavidin labelled by means of phosphorylation may be used directlyto enhance immunoassays as a substitute for unlabelled streptavidin orenzyme-linked unlabelled streptavidin. The invention also contemplatesintroducing phosphorus or analog labels into genetically engineeredantibodies (see references 93-99), more particularly Mabs, or in the Fabor Fab′ fragment. Such Mabs are useful for diagnostic and therapeuticpurposes. The phosphorylated Mabs can be made to target specifictumor-associated antigens or a variety of tumors, like breast and coloncancer cells, malignant melanoma cells, ovarian carcinoma cells, andother malignant tumors.

6. Further Therapeutic Uses.

Other uses contemplated in accordance with the invention are as follows:Monoclonal or appropriate cocktails of antibodies and/or antibodyfragments (such as the Fab or Fab′ fragments) are fruitful molecules inwhich in accordance with the invention phosphorylation or otherlabellable sites can be introduced. The use of ³²P in therapy has beendemonstrated f or polycythemia vera and other malignancies (116). Thus,it is clear that the high energy β particle is effective as ananticellular agent. The attachment of ³²P through the introduction ofphosphorylation site (s) in Mabs or their appropriate fragments (Fab andFab′) would also be effective for the therapy of tumors to which thesemonoclonal antibodies are specific. A large number of monoclonalantibodies have been developed to tumor-associated antigens from breast,colon, ovarian, and other adenocarcinomas, malignant melanoma, and manyother tumors. Thus, Mabs directed to the tumor associated antigens ofthese tumors are expected to be highly effective when labelled with ³²P.The labelling can be increased by use of cassettes of phosphorylationsites or directly by introduction of multiple phosphorylation sites intothe intact protein or the appropriate fragments through geneticengineering. By “cassette” is meant a multifunctional moiety.

When multiple labelled phosphorylation sites are introduced inaccordance with the invention in Mabs, this may reduce the bindingspecificity and/or affinity of the modified Mabs for the specificepitope targeted. It can be seen that under such circumstances the useof a biotinylated Mab linked to the multiple phosphorylated streptavidin(as described above) has distinct advantages; the specificity of the Mabis not altered and yet the radioactivity of the diagnostic agent hasbeen many-fold enhanced.

The invention also has implications for the preparation of therapeuticagents to which patients are likely to develop an adverse antigenicresponse. Thus, the monoclonal antibodies can be engineered successivelyin accordance with the invention with different phosphorylation sites.When introduced into patients who have become sensitive to or who areproducing antibodies to the injected antibody because of thephosphorylation site, then by changing to a different phosphorylationsite, the antigenic character of the protein can be modified. Thus, itmay be possible to use such antibodies in multiple successivetherapeutic regimens in patients who are reacting with the antibody ofthe previous type. For this purpose a series of antibodies with avariety of phosphorylation sites can be developed. Each series would bedesigned to have a different epitopic structure and be usedsequentially. Alternatively a cocktail of such different antibodies canbe used initially so that any one is present at a fraction of the total.This would minimize antibody formation to any one of the new sites.

7. Various Isotopes.

In accordance with the invention, as discussed above, phosphorylatedderivatives should be serum-stable for certain applications. Variousisotopes can be employed that are more effective than others for aspecific therapeutic purpose. For example, ³³P may be substituted for³²P in the phosphorylation reaction. It is less likely that ³⁵S with ahalf-life of about 89 days would be normally as useful as ananticellular reagent because it is a low energy β emitter. Nevertheless,conceivably there may be specific uses for ³⁵S labelled monoclonalantibodies in therapy and/or diagnosis.

Table 3 below shows various isotopes (and other pertinent particulars)which are especially useful for introduction into proteins in accordancewith the invention.

TABLE 3 Isotopes for Labellable Groups Isotope Half-Life Type of DecayEnergy of Radiation ^(32P) 14.2 days β− 1.707 Mev ³³P 24.4 days β− 0.25Mev ³⁵S 87.0 days β− 0.167 Mev ³⁸S 2.87 hours β− 1.1 Mev

Decay factors and radioactivity at any given time is available in theliterature. For instance, for comparison between ¹²⁵I and ¹³¹I with, onthe other hand, ³²P and ³⁵S, reference is made to Appendices, TableA.1.7 (Supplement 2) in Current Protocols in Molecular Biology, cited(43).

Thus, the invention provides tailored-designed proteins for specificbiological purposes.

An important implication of this invention is the greater safety of thelabelled Mabs due to lower energy emission levels and the nature of theradio emission. Specifically, Mabs labelled with ³²P or ³³P havesignificantly lower energy emission levels than conventionalradio-labels for protein such as ¹²⁵I; moreover, the decay emission ofthe phosphorus and sulfur isotopes (³²P, ³³P, ³³P and ³⁸S) is betaparticles, as compared to gamma rays of ¹²⁵I as are common in existinglabelling protocols.

The safety feature of the beta-labelled proteins, e.g., Mabs orstreptavidins (as discussed) in accordance with the invention, is verysignificant for diagnostic and therapeutic uses of the invention. Betaemitters penetrate the tumor but are not emitted as readily as gamma rayemitters from the patient to surrounding medical staff and non-medicalattending individuals.

By selecting ³⁵S (which has a half-life of 87 days) and the ³⁵Sphosphate ATP analog to ³²P one can significantly increase the effectiveradioactive life of the therapeutic agent.

Thus, the proteins labelled in accordance with the invention have aspectrum of meaningful advantageous properties heretofore not readilyavailable.

The invention is not limited to the use of unstable isotopes. In thefuture it may be advantageous to label a protein with a stable isotopethat would be suitable for detection by NMR, nuclear activation, orfuture developed procedures. Nor is it necessary that the label be a“radio” label providing it is an identifiable label.

8. Radioimmunoassays with Labelled Antigens.

In accordance with the invention the phosphorylated proteins can begenerally used as the radio-labelled component. These radioimmunoassayscan be used with polyclonal as well as with monoclonal antibodies. Ifthe introduction of a new phosphorylation site into a protein changesthe antigenic structure of the protein in the area of thephosphorylation site, or even at distant linear positions of theprotein, and alters the antigenic behavior, the protein in accordancewith the invention, can be modified to introduce a phosphorylation siteat a different position so that the antigenic behavior will remainstable and for the protein to bind with the polyclonal or monoclonalantibody of interest.

Thus, the invention provides considerable versatility regarding theposition where the label can be introduced. Generally it will bepreferred to introduce the phosphorus (or other radio-label) at a sitethat will not disrupt the antigen-antibody binding.

9. Sandwich Radioimmunoassays.

In sandwich radioimmunoassays with monoclonal antibodies, theintroduction of phosphorylation sites into an antibody in accordancewith the invention is a sensitive method to follow the binding of thesecond antibody. Thus, the sensitivity of such sandwichradioimmunoassays can be increased substantially. Particularly, whenmultiple phosphorylation sites are introduced in accordance with theinvention into the protein directly or by the addition of a fusionphosphorylation cassette; the sensitivity of such assays will beincreased many-fold.

Another advantage of the invention is to be noted. Because thephosphorylation reaction is gentle, unlike the iodination or otherchemical modifications necessary to radio-label proteins with iodine orother reagents, monoclonal antibodies that are inactivated by thechemical or iodination procedures are not likely to be inactivated bythe phosphorylation procedure. Thus, the process of the invention allowsfor the phosphorylation of proteins normally too sensitive for labellingwith iodine. The introduction of a phosphate analog with ³⁵S provides aradio-labelled protein derivative with a long half-life (1.5 timeslonger than ¹²⁵I and 6 times longer than ³²P). Thus, when Mabs arelabelled with ³⁵S, they will have a substantially longer shelf-lifecompared to the ³²P or ¹²⁵I radio-labelled derivatives.

As discussed above, the invention allows for the selection of the mostappropriate labelling isotope, as compared to ¹²⁵I, for instance.

10. Imaging.

Generally for imaging of tumors or tissues in an animal or a patient, ahigh energy gamma emitter is generally preferable to a high energy βemitter, which by and large would be absorbed by the tissues. However,in certain imaging studies in animals or in patients, Mabs to which ³²P,³³P or ³⁵S are attached through introduced phosphorylation sites inaccordance with the invention may be useful.

For example, it can be seen that Mabs labelled with ³²P, ³³P or ³⁵Scould be useful in in vivo studies in which biopsy specimens are to beexamined. The spread of a tumor during surgery could be followed byutilizing a radioisotope detector probe to follow the local spread ofthe tumor and guide the extent of the surgery. In addition, tissuespecimens which are fixed or frozen can be taken to which these proteinswill remain bound (that is, antibodies to the tumor-associated antigensor other ligands). Thus, autoradiographs of tissue sections can provideinformation about the extent of tumor spread and the extent of bindingof specific monoclonal antibodies to tumor-associated antigens can bethoroughly evaluated. Furthermore, as an in vitro reagent with cells ortissue slices, such labelled antibodies would be highly sensitivereagents to detect tumor-associated antigens or other antigens by theusual types of assays employed.

11. Anti-antibodies.

There are many known uses for anti-antibodies such as anti-mouse,anti-human, anti-sheep, and anti-goat antibodies, etc. or monoclonalantibodies as single entities or as a cocktail. Such antibodies can beengineered in accordance with the invention to introduce single ormultiple phosphorylation sites and, accordingly labelled with a varietyof isotopes as described above. These provide general reagents whereanti-antibodies are necessary, particularly in radioimmunoassays,autoradiography, or any other reactions in which anti-antibodies areuseful.

12. Rapid Purification of Phosphorylated Proteins.

The invention has also applications in separating and purifyingproteins. Proteins which are phosphorylated can be separated from thosewhich are not; proteins which are more phosphorylated than others can beseparated.

For instance, where proteins can be phosphorylated, it is common foronly a percentage of the molecules to be phosphorylated. The totalphosphorylation, of course, can be enhanced by the introduction ofmultiple phosphorylation sites in the protein in accordance with theinvention so that few molecules escape phosphorylation. To be able toseparate the phosphorylated from the non-phosphorylated proteins isespecially useful for molecules with a single phosphorylation site wherethere may be phosphorylated and non-phosphorylated molecules in thepopulation. In this manner, the effectiveness of any phosphorylatedderivatives is increased. Separation of phosphorylated fromnon-phosphorylated molecules can be accomplished by developingpolyclonal or monoclonal antibodies to the phosphorylation sites withand/or without derivatized phosphate groups. Such polyclonal andmonoclonal antibodies are expected to have considerable value inpurifying the proteins and have been described (see for example119-124).

13. Dephosphorylation of Proteins.

Considerable emphasis has been, placed herein on aspects ofphosphorylation. It is a consequence of the phosphorylation (withphosphate or thiophosphate groups) that the removal of the label is alsofacilitated in that dephosphorylation is a milder procedure which tendsto be less disruptive of the protein molecule than procedures in theprior art for removal of ¹²⁵I from proteins. Thus, in cases where it isuseful to remove the radioisotope, this can be achieved relativelyeasily and gently by an enzyme reaction. A variety of phosphatases canbe used for this purpose. Most phosphatases have comparatively lowspecificity (for example, reference 100, pages 192-193, 203, 223-224,736-739) although a few have very high specificity such as those actingon sugar phosphates and the enzyme that dephosphorylates glycogensynthetase b and phosphorylase b (47, 100; also reference 101 pages372-373, for example). Furthermore, specific dephosphorylation ofphosphorylated proteins can be achieved by reversal of the reaction ofprotein-serine and -tyrosine kinases (107). If it is necessary todetermine whether in fact the phosphate addition causes a change in theactivity of the protein, rather than aging, denaturation, or othermanipulations, the phosphate can be removed and the activity of theprotein again determined. In such a manner, a definitive understandingof the effect of phosphorylation on the activity of the protein can beassessed. This may be useful in determining the activities of variousphosphorylated interferons.

The concept of “dephosphorylation” has an interesting application whichis essentially the “converse” of that taught herein. Wherever a site ina protein in the native state is naturally phosphorylatable the removalof that site would be particularly desirable when it is known that thenaturally phosphorylatable protein causes some undesired results. Anillustration would be proteins associated with oncogenic viruses such asRous sarcoma virus (RSV) and cellular oncogenes.

14. Phosphorylation Cassettes.

The invention also contemplates an alternative method for labellingproteins without inserting the coding sequence for the phosphorylationsite (or cassette) into the nucleotide coding sequence of the protein,and yet still use the invention. This procedure would be particularlyuseful for large proteins like immunoglobulins for use in variousassays. Such alternative method calls for a polypeptide which isphosphorylated to be chemically linked to the large protein. The linkingwould be by any bifunctional reagent or an activated derivative (likeN-hydroxy-succinimide), as is known in the art.

This technique could use a polypeptide with multiple phosphorylationsites in tandem or “cassette” that can be introduced within or at eitherend of a protein. The DNA coding for the tandem phosphorylation siteswould be flanked by restriction sites for easy cleaving and insertioninto the DNA containing the coding sequence for the protein to be linkedto the larger protein. Such a phosphorylation cassette could beexpressed as a small polypeptide then phosphorylated and then chemicallylinked to the larger protein.

15. Phosphorylatable Human or Animal Donor Genes.

Further, it is within the contemplation of the invention to provide DNAsequences engineered into appropriate vectors or cell lines or even intoanimals by transgenic techniques. Thus cells or animals could producephosphorylatable (and/or phosphorylated) proteins such asimmunoglobulins after phsphorylation sites are introduced into theproteins by the methods of this invention. Phosphorylatable chimericantibodies with a mouse variable region and human constant region couldbe developed (93-99). The human antibodies used as the donor moleculewould be engineered to contain single or multiple phosphorylation sites.Analogously, this could be applied to proteins other thanimmunoglobulins.

16. Use of Phosphorylation Sites to Map Tertiary Structure of Proteins.

By introducing a small phosphorylation recognition site into a proteinrandomly along the entire linear protein chain, it will be possible toobtain information about the tertiary structure of proteins. Thesequence encoding the phosphorylation site is inserted randomly withinthe DNA sequence encoding the protein of interest. The insertion must bemade in such a way that the phosphorylation sequence is in phase withupstream and downstream codons so that an insertion for aphosphorylation site is made without interrupting the phase oftranslation. The expressed protein, therefore, contains the identicallinear sequence of the original protein with an insertion of aphosphorylation site in a given position along the chain. By generatinga large series of insertions (ideally after every amino acid position ofthe protein chain), it is possible to determine whether the kinaserecognizes the sequence in the context in which it is placed by a simpleassay to determine the rate and extent of phosphorylation at thatposition. The rate and extent of phosphorylation depends on theaccessibility of that site to the phosphokinase which reflects itsposition in the tertiary structure (outside, internal, buried, etc.). Acomplete linear map of the accessibility of the phosphorylation sitesalong the entire chain will provide an outline of the structuralfeatures of the protein that are inside and outside in the tertiarystructural configuration. Insertions of amino acids should be designedto minimize perturbations. In some cases insertion of a phosphorylationsite can occur by simply changing one or more amino acids rather thaninserting several amino acids comprising the phosphorylation recognitionsite.

The generation of such DNA insertions to make the appropriate variety ofinsertional mutant proteins can be done in many ways. Insertions can beintroduced along a protein chain systematically or randomly by methodscomparable to saturation mutagenesis. Alternatively, rather thangenerating mutants from a given DNA sequence by inserting the sequenceencoding the phosphorylation site into the DNA, one can generatesynthetic oligonucleotides so that the entire DNA chain is synthesizedde novo. Combinations of these procedures and general cloning strategiescould easily provide an entire bank of new mutant proteins withphosphorylation sites distributed linearly along the chain.

This procedure provides information about the tertiary structure andfolding of the protein in solution. It compliments methods such as x-raycrystallography which will provide tertiary structure information of theproteins in the crystal. Furthermore, the method will be useful todetermine the tertiary structure of proteins which have resisted effortsto obtain appropriate crystals for determination of X-raycrystallographic structures.

This aspect is an illustration of a protein having numerous putativephosphorylation sites, ideally after each amino acid in the sequence ofthe protein; and the corresponding phosphorylated protein. Likewise,this is an illustration of a DNA sequence encoding the putativephosphorylation site(s) inserted in the DNA sequence encoding theselected protein of interest.

17. Other Applications.

There are other applications for the labelled proteins of the invention.In general virtually any protein that contains a label (radio-label,fluorescent-label, chemical-label, enzyme-label, etc.) can alternativelybe labelled with phosphate by the introduction of phosphorylationsite(s) in accordance with the invention. The purification of suchproteins can be followed in a sensitive assay by simply measuring theability to accept a phosphate group rather than to follow enzymeactivity. Such proteins engineered in accordance with the invention,therefore, can be purified easily and themselves be used as a tracer tofollow the purification of other proteins to which they are similar. Forexample, it is likely that a protein with a single phosphorylation siteengineered with very little modification of the protein structure itselfwould be purified similarly to the unmodified protein.

In practice, by having a stock of phosphorylatable proteins or series ofmarkers, the labelled derivatives can be prepared conveniently by thesimple phosphorylation reaction when desired. Thus, the proteins of theinvention which are phosphorylatable provide a useful inventory of thecorresponding labelled proteins.

18. Pharmaceutical and Biologically Active Compositions.

The modified proteins of the invention can be formulated according toknown methods to prepare pharmaceutically useful compositions. Forinstance, the human alpha interferon-like protein hereof is combined inadmixture with a pharmaceutically acceptable carrier vehicle. Suitablevehicles and their formulation are described in Remington'sPharmaceutical Sciences by E. W. Martin, which is hereby incorporated byreference. Such compositions will contain an effective amount of theinterferon-like protein or other proteins hereof together with asuitable amount of vehicle in order to prepare pharmaceuticallyacceptable compositions suitable for effective administration to thehost. The host can be a mammal or not. The carrier may be liquid, solid,or other. Of course therapeutic applications for humans and veterinaryapplications are intended for the biologically active compositions ofthe invention. The biologically active composition of the invention isto be administered in a biologically or therapeutically effective amountwhich can be readily determined by one skilled in the art. Generally itis the smallest amount for which a desired response will be obtained toan amount which is excessive for practical or other purposes.

The biologically active compositions of the invention can also includeany other biologically active substance which does not adversely affectthe desired activity, particularly the activity or use of the modifiedprotein of the invention.

It is understood that the modified proteins of the invention can beobtained by chemical and/or enzymatic synthesis rather than byrecombinant DNA technology.

While reference has been made to particular preferred embodiments and toseveral uses and applications made possible by the invention, it will beunderstood that the present invention is not to be construed as limitedto such, but rather to the lawful scope of the appended claims andsubject matter covered by the doctrine of equivalents.

From the description provided hereinabove it will be appreciated by oneskilled in the art that the invention makes a significant andmeritorious contribution to the art.

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What is claimed is:
 1. A recombinant expression vector for expressing anexogenous functional mammalian protein not normally phosphorylated invivo, which vector comprises (i) a first coding sequence for aphosphorylation recognition sequence for a eukaryotic protein kinase,(ii) a restriction site for adding a second coding sequence for apolypeptide sequence of interest in the same reading frame as thephosphorylation recognition sequence such that the vector, upon additionof said coding sequence, encodes a fusion protein comprising both saidphosphorylation recognition sequence and said polypeptide sequence ofinterest, and (iii) a promoter sequence for regulating expression ofsaid fusion protein in a host cell.
 2. The vector of claim 1, whichfurther comprises an origin of replication for replicating the vector ina host cell.
 3. The vector of claim 2, wherein the origin of replicationis a bacterial origin of replication.
 4. The vector of claim 1, whichfurther comprises a marker gene for selecting host cells transformedwith said vector.
 5. The vector of claim 4, wherein the marker geneconfers antibiotic resistance to the host cell.
 6. The vector of claim1, which further comprises a polylinker sequence for cleaving the vectorwith one or more restriction enzymes and ligating the first codingsequence into the reading from of said phosphorylation recognitionsequence.
 7. The vector of any of claims 1, 2, 4 or 6, wherein thephosphorylation recognition sequence is a recognition sequence for aserine/threonine kinase.
 8. The vector of any of claims 1, 2, 4 or 6,wherein the phosphorylation recognition sequence is a recognitionsequence for a tyrosine kinase.
 9. The vector of any of claims 1, 2, 4or 6, wherein the phosphorylation recognition sequence is a recognitionsequence for a cyclic-AMP-dependent protein kinase.
 10. The vector ofany of claims 1, 2, 4 or 6, wherein the phosphorylation recognitionsequence is a recognition sequence for cyclic GMP-dependent proteinkinase.
 11. The vector of any of claims 1, 2, 4 or 6, wherein thephosphorylation recognition sequence is a recognition sequence for acasein kinase.
 12. The vector of claim 1, wherein the phosphorylationrecognition sequence comprises the sequence Arg-Arg-Xaa-Ser-Xaa.
 13. Thevector of claim 1 wherein the protein is selected from the groupconsisting of hormones, cytokines, lymphokines and growth factors.